ABSTRACTS
of CURRENT and RECENT PAPERS
Biochemistry 2006 45(26), 8135-42
Zhu M, Qin ZJ, Hu D, Munishkina
LA, Fink AL
Alpha-synuclein,
a presynaptic protein associated with Parkinson's disease, is found as both
soluble cytosolic and membrane-bound forms. Although the function of
alpha-synuclein is unknown, several observations suggest that its association
with membranes is important. In the present study we investigated the effect of
alpha-synuclein on lipid oxidation in membranes containing phospholipids with
unsaturated fatty acids. The kinetics of lipid oxidation were monitored by the
change in fluorescence intensity of the dye C11-BODIPY. We find that monomeric
alpha-synuclein efficiently prevented lipid oxidation, whereas fibrillar
alpha-synuclein had no such effect. Our data suggest that the prevention of
unsaturated lipid oxidation by alpha-synuclein requires that it bind to the
lipid membrane. The antioxidant function of alpha-synuclein is attributed to
its facile oxidation via the formation of methionine sulfoxide, as shown by
mass spectrometry. These findings suggest that the inhibition of lipid
oxidation by alpha-synuclein may be a physiological function of the protein.
Fibrillation of
human insulin A and B chains.
Biochemistry 2006
Aug 1; 45(30), 9342-53
Hong, DP, Ahmad, A., Fink AL
Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions. We examined whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH. Although insulin exhibits a pH-dependent lag phase in fibrillation, the A chain formed fibrils without a lag at both pHs. In contrast, the B chain exhibited complex concentration-dependent fibrillation behavior at acidic pH. At higher concentrations, e.g., >0.2 mg/mL, the B chains preferentially and rapidly formed stable protofilaments rather than mature fibrils upon incubation at 37 degrees C. Surprisingly, these protofilaments did not convert into mature fibrils. At lower B chain concentrations, however, mature fibrils were formed. The explanation for the concentration dependence of B chain fibrillation is as follows. The B chains exist as soluble oligomers at acidic pH, have a beta-sheet rich conformation as determined by CD, and bind ANS strongly, and these oligomers rapidly form dead-end protofilaments. However, under conditions in which the B chain monomer is present, such as low B chain concentration (<0.2 mg/mL) or in the presence of low concentrations of GuHCl, which dissociates the soluble oligomers, mature fibrils were formed. Thus, both A and B chain peptides can form amyloid fibrils, and both are likely to be involved in the interactions leading to the fibrillation of intact insulin.
Independent
Heterologous Fibrillation of Insulin and its B-chain Peptide
Biochemistry
2005,44, 16701-16709
Dong-Pyo Hong, and Anthony L Fink*
Insulin is very prone to form amyloid fibrils under slightly destabilizing conditions, and the B-chain region plays a critical role in the fibrillation. We show here that the isolated B-chain peptide of bovine insulin also forms fibrils at both acidic and neutral pH. When a mixture of insulin and the B-chain peptide was incubated at either acidic or neutral pH, the formation of fibrils was clearly separated into two phases, the faster corresponding to fibrils from the B-chain and the slower to fibrillation of insulin. To further investigate the interaction (or lack thereof) between the two polypeptides, we examined the effects of cross-seeding. The results indicate that seeds of B-chain fibrils accelerate the fibrillation of insulin at pH 1.6, and inhibit the fibrillation at pH 7.5, but seeds of insulin fibrils have little effect on the fibrillation of the B-chain. Our results demonstrate that in the co-aggregation of closely related peptides each peptide species may undergo concurrent homogeneous or heterologous polymerization, and that fibrils of one species may or may not seed fibrillation of the other. The results demonstrate the significant “species” barrier in amyloid fibril formation between fibrillation induced by different fibrils. A model for the fibrillation of the heterogeneous system of insulin and B-chain insulin is proposed.
Concerted Action of Metals and Macromolecular Crowding on
theFibrillation of α-Synuclein
Larissa A. Munishkina,
Vladimir N. Uversky and Anthony L. Fink*
The Oxidation State Of DJ-1 Regulates Its Chaperone Activity
Toward α-Synuclein
Wenbo
Zhou, BS; Min Zhu, PhD; Mark A Wilson, PhD; Gregory A Petsko, PhD; Anthony L
Fink, PhD
Abstract: DJ-1 has been reported to have chaperone activity by preventing
the aggregation of some proteins, and by structural analogy to Hsp31. The L166P
mutation has been linked to a familial early onset form of Parkinson's disease.
Since the aggregation of alpha-synuclein is believed to be a critical step in
the etiology of PD, we have investigated the interaction of wild-type DJ-1 and
its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not
inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was
observed. However, DJ-1 is very susceptible to oxidation by the addition of two
oxygens to form the sulfinic acid of Cys 106 (2O DJ-1) (no 1O oxidized state is
detectable). 2O DJ-1 was readily prepared by the addition of H2O2 at
concentrations up to a 20-fold molar excess. The oxidation of Cys 106 to the
sulfinic acid had minimal effect on the structural properties of DJ-1. However,
2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and
only this form of DJ-1 appears to have significant anti-aggregation properties
against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some
secondary structure, and to loss of the ability to inhibit alpha-synuclein
fibrillation. Our observations confirm the suggestion that DJ-1 may act as an
oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation.
Since oxidative stress has been associated with PD, this observation may
explain why mutations of DJ-1 could be a contributing factor in PD, and also
indicates that excess oxidative stress could also lead to enhanced
alpha-synuclein aggregation and hence PD.
Journal Biological Chemistry 2005 Dec. 30, 280:52,
42669-42675
Atta Ahmad, Vladimir N. Uversky, Dongpyo Hong and Anthony L. Fink*
Insulin has a
largely α-helical structure, and
exists as a mixture of hexameric, dimeric and monomeric states in solution,
depending on the conditions: the protein is monomeric in 20% acetic acid.
Insulin forms amyloidlike fibrils under a variety of conditions, especially at
low pH. In this study we investigated the fibrillation of monomeric human
insulin by monitoring changes in CD, ATR-FTIR, ANS fluorescence, ThT
fluorescence, dynamic light scattering and H/D exchange during the initial
stages of the fibrillation process in order to provide insight into early
events involving the monomer. The results demonstrate the existence of
structural changes occurring before the onset of fibril formation, which are
detectable by multiple probes. The data indicate at least two major populations
of oligomeric intermediates between the native monomer and fibrils. Both have
significantly non-native conformations, and indicate that fibrillation occurs
from a betarich structure significantly distinct from the native fold.
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J Mol Biol.
2005 Oct 21;353(2):357-72 |
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Kaylor J, Bodner N, Edridge S, Yamin G, Hong DP, Fink AL.
The aggregation of alpha-synuclein is believed to be a
critical step in the etiology of Parkinson's disease. A variety of biophysical
techniques were used to investigate the aggregation and fibrillation of
alpha-synuclein in which one of the four intrinsic Tyr residues was replaced by
Trp, and two others by Phe, in order to permit fluorescence resonance energy
transfer (FRET) between residues 39 (Tyr) and 125 (Trp). The mutant
Y125W/Y133F/Y136F alpha-synuclein (one Tyr, one Trp) showed fibrillation
kinetics similar to that of the wild-type, as did the Y125F/Y133F/Y136F (one
Tyr, no Trp) and Y39F/Y125W/Y133F/Y136F (no Tyr, one Trp) mutants. Time-dependent
changes in FRET, Fourier transform infrared, Trp fluorescence, dynamic
light-scattering and other probes, indicate the existence of a transient
oligomer, whose population reaches a maximum at the end of the lag time. This
oligomer, in which the alpha-synuclein is in a partially folded conformation,
is subsequently converted into fibrils, and has physical properties that are
distinct from those of the monomer and fibrils. In addition, another population
of soluble oligomers was observed to coexist with fibrils at completion of the
reaction. The average distance between Tyr39 and Trp125 decreases from 24.9A in
the monomer to 21.9A in the early oligomer and 18.8A in the late oligomer.
Trp125 remains solvent-exposed in both the oligomers and fibrils, indicating
that the C-terminal domain is not part of the fibril core. No FRET was observed
in the fibrils, due to quenching of Tyr39 fluorescence in the fibril core.
Thus, aggregation of alpha-synuclein involves multiple oligomeric intermediates
and competing pathways.
Agrin Binds a-Synuclein
and Modulates a-Synuclein Fibrillation.
Glycobiology.
2005 15:12, 1320-1331
Liu IH, Uversky VN, Munishkina LA,
Fink AL, Halfter W, Cole GJ.
Recent studies have
begun to investigate the role of agrin in brain, and suggest that agrin's
function likely extends beyond that of a synaptogenic protein. Particularly it
has been shown that agrin is associated with the pathological lesions of
Alzheimer's disease (AD) and may contribute to the formation of ss-amyloid
plaques in AD. We have extended the analysis of agrin's function in
neurodegenerative diseases to investigate its role in Parkinson's disease (PD).
alpha-Synuclein is a critical molecular determinant in familial and sporadic
PD, with the formation of alpha-synuclein fibrils being enhanced by sulfated
macromolecules. In the studies reported here we show that agrin binds to
alpha-synuclein in a heparan sulfate-dependent manner , induces conformational
changes in this protein characterized by ss-sheet structure, and enhances
insolubility of alpha-synuclein. We also show that agrin accelerates the formation
of protofibrils by alpha-synuclein, and decreases the half-time of fibril
formation. The association of agrin with PD lesions was also explored in PD
human brain, and these studies shown that agrin co-localizes with
alpha-synuclein in neuronal Lewy bodies in the substantia nigra of PD brain.
These studies indicate that agrin is capable of accelerating the formation of
insoluble protein fibrils in a second common neurodegenerative disease. These
findings may indicate shared molecular mechanisms leading to the
pathophysiology in these two neurodegenerative disorders.
Forcing
non-amyloidogenic beta-synuclein to fibrillate.
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Biochemistry.
2005 Jun 28;44(25):9096-107. |
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Yamin G, Munishkina LA,
Karymov MA, Lyubchenko YL,
Uversky VN, Fink AL.
The fibrillation and
aggregation of alpha-synuclein is a key process in the formation of
intracellular inclusions, Lewy bodies, in substantia nigral neurons and,
potentially, in the pathology of Parkinson's disease and several other
neurodegenerative disorders. Alpha-synuclein and its homologue beta-synuclein
are both natively unfolded proteins that colocalize in presynaptic terminals of
neurons in many regions of the brain, including those of dopamine-producing
cells of the substantia nigra. Unlike its homologue, beta-synuclein does not
form fibrils and has been shown to inhibit the fibrillation of alpha-synuclein.
In this study, we demonstrate that fast and efficient aggregation and
fibrillation of beta-synuclein can be induced in the presence of a variety of
factors. Certain metals (Zn(2+), Pb(2+), and Cu(2+)) induce a partially folded
conformation of beta-synuclein that triggers rapid fibrillation. In the
presence of these metals, mixtures of alpha- and beta-synucleins exhibited
rapid fibrillation. The metal-induced fibrillation of beta-synuclein was
further accelerated by the addition of glycosaminoglycans or high
concentrations of macromolecular crowding agents. Beta-synuclein also rapidly
formed soluble oligomers and fibrils in the presence of pesticides, whereas the
addition of low concentrations of organic solvents induced formation of
amorphous aggregates. These new findings demonstrate the potential effect of
environmental pollutants in generating an amyloidogenic, and potentially
neurotoxic, conformation, in an otherwise benign protein.
Effects of nitration on the structure and
aggregation of alpha-synuclein.
Brain Res Mol Brain Res. 2005 Mar
24;134(1):84-102.
Uversky
VN, Yamin G, Munishkina LA, Karymov MA, Millett IS, Doniach S, Lyubchenko YL,
Fink AL.
Substantial evidence suggests that the
aggregation of the presynaptic protein alpha-synuclein is a key step in the
etiology of Parkinson's disease (PD). Although the molecular mechanisms underlying
alpha-synuclein aggregation remain unknown, oxidative stress has been
implicated in the pathogenesis of PD. Here, we report the effects of tyrosine
nitration on the propensity of human recombinant alpha-synuclein to fibrillate
in vitro. The properties of nitrated alpha-synuclein were investigated using a
variety of biophysical and biochemical techniques, which revealed that
nitration led to formation of a partially folded conformation with increased
secondary structure relative to the intrinsically disordered structure of the
monomer, and to oligomerization at neutral pH. The degree of self-association
was concentration-dependent, but at 1 mg/mL, nitrated alpha-synuclein was
predominantly an octamer. At low pH, small-angle X-ray scattering data indicated
that the nitrated protein was monomeric. alpha-Synuclein fibrillation at
neutral pH was completely inhibited by nitrotyrosination and is attributed to
the formation of stable soluble oligomers. The presence of heparin or metals
did not overcome the inhibition; however, the inhibitory effect was eliminated
at low pH. The addition of nitrated alpha-synuclein inhibited fibrillation of
non-modified alpha-synuclein at neutral pH. Potential implications of these
findings to the etiology of Parkinson's disease are discussed.
Current Opinion in Structural
Biology Volume 15, Issue 1,
February 2005, Pages 35-41
Anthony L Fink
It is now clear that a significant fraction of eukaryotic genomes encode proteins with substantial regions of disordered structure. In spite of the lack of structure, these proteins nevertheless are functional; many are involved in critical steps of the cell cycle and regulatory processes. In general, intrinsically disordered proteins interact with a target ligand (often DNA) and undergo a structural transition to a folded form when bound. Several features of intrinsically disordered proteins make them well suited to interacting with multiple targets and to cell regulation. New algorithms have been developed to identify disordered regions of proteins and have demonstrated their presence in cancer-associated proteins and proteins regulated by phosphorylation.
Methionine oxidation, α-synuclein and
Parkinson's disease
Biochimica et Biophysica Acta (BBA) - Proteins &
Proteomics
Volume 1703, Issue 2 , 17 January 2005, Pages
157-169
Methionine Oxidation and Methionine Sulfoxide Reductases
Charles B. Glaser, Ghiam Yamin, Vladimir N. Uversky and
Anthony L. Fink
The aggregation of normally soluble α-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson's disease. Oxidative stress is believed to be a contributing factor in this disorder. Because it lacks Trp and Cys residues, mild oxidation of α-synuclein in vitro with hydrogen peroxide selectively converts all four methionine residues to the corresponding sulfoxides. Both oxidized and non-oxidized α-synucleins have similar unfolded conformations; however, the fibrillation of α-synuclein at physiological pH is completely inhibited by methionine oxidation. The inhibition results from stabilization of soluble oligomers of Met-oxidized α-synuclein. Furthermore, the Met-oxidized protein also inhibits fibrillation of unmodified α-synuclein. The degree of inhibition of fibrillation by Met-oxidized α-synuclein is proportional to the number of oxidized methionines. However, the presence of metals can completely overcome the inhibition of fibrillation of the Met-oxidized α-synuclein. Since oligomers of aggregated α-synuclein may be cytotoxic, these findings indicate that both oxidative stress and environmental metal pollution could play an important role in the aggregation of α-synuclein, and hence possibly Parkinson's disease. In addition, if the level of Met-oxidized α-synuclein was under the control of methionine sulfoxide reductase (Msr), then this could also be factor in the disease.
Role of Protein-Water Interactions and Electrostatics in
a-Synuclein
Fibril Formation
Biochemistry,
Rifampicin
Inhibits alpha-Synuclein Fibrillation and Disaggregates Fibrils.
Chem Biol. 2004 Nov;11(11):1513-21.
Li J, Zhu M, Rajamani S, Uversky VN, Fink AL.
The aggregation of
alpha-synuclein in dopaminergic neurons of the substantia nigra is a critical
step in the pathogenesis of Parkinson's disease. We show that the antibiotic
rifampicin inhibited alpha-synuclein fibrillation and disaggregated existing
fibrils in a concentration-dependent manner. Size-exclusion chromatography data
indicated that rifampicin stabilized alpha-synuclein as both a monomer and
soluble oligomers comprised of partially folded alpha-synuclein. Experiments
using aged samples of rifampicin indicated that the most active species in
inhibiting fibrillation and disaggregating fibrils is an oxidation product of
rifampicin, which was confirmed in experiments under anaerobic conditions.
These results indicate that rifampicin-mediated inhibition of alpha-synuclein
fibrillation and disaggregation of fibrils involves preferential stabilization
of monomeric and soluble oligomeric forms, and that rifampicin potentially may
have therapeutic application for Parkinson's disease.
The
effect of macromolecular crowding on protein aggregation and amyloid fibril
formation
Munishkina, L.A.,
Cooper, E.M., Uversky, V.N., and Fink, A.L.
Macromolecular crowding is expected to have several significant effects on protein aggregation; the major effects will be those due to excluded volume and increased viscosity. In this report we summarize data demonstrating that macromolecular crowding may lead to a dramatic acceleration in the rate of protein aggregation and formation of amyloid fibrils, using the protein α-synuclein. The aggregation of α-synuclein has been implicated as a critical factor in development of Parkinson’s disease. Various types of polymers, from neutral polyethylene glycols and polysaccharides (Ficolls, dextrans) to inert proteins, are shown to accelerate α-synuclein fibrillation. The stimulation of fibrillation increases with increasing length of polymer, as well as increasing polymer concentration. At lower polymer concentrations (typically up to ~100 mg/ml) the major effect is ascribed to excluded volume, whereas at higher polymer concentrations evidence of opposing viscosity effects become apparent. Pesticides and metals, which are linked to increased risk of Parkinson’s disease by epidemiological studies, are shown to accelerate α-synuclein fibrillation under conditions of molecular crowding.
Conformational prerequisites for formation of amyloid fibrils from histones.
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J Mol Biol. 2004 Sep 24;342(4):1305-24. |
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Munishkina LA, Fink AL, Uversky
VN.
We demonstrate that bovine core histones are
natively unfolded proteins in solutions with low ionic strength due to their
high net positive charge at pH 7.5. Using a variety of biophysical techniques
we characterized their conformation as a function of pH and ionic strength, as
well as correlating the conformation with aggregation and amyloid fibril
formation. Tertiary structure was absent under all conditions except at pH 7.5
and high ionic strength. The addition of trifluoroethanol or high ionic
strength induced significant alpha-helical secondary structure at pH 7.5. At
low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC
indicate the histones are present as a hexadecamer of globular subunits. The
secondary structure at low pH was independent of the ionic strength or presence
of TFE, as judged by FTIR. The data indicate that histones are able to adopt
five different relatively stable conformations; this conformational variability
probably reflects, in part, their intrinsically disordered structure. Under
most of the conditions studied the histones formed amyloid fibrils with typical
morphology as seen by electron microscopy. In contrast to most
aggregation/amyloidogenic systems, the kinetics of fibrillation showed an
inverse dependence on histone concentration; we attribute this to partitioning
to a faster pathway leading to non-fibrillar self-associated aggregates at
higher protein concentrations. The rate of fibril formation was maximal at low pH,
and decreased to zero by pH 10. The kinetics of fibrillation were very
dependent on the ionic strength, increasing with increasing salt concentration,
and showing marked dependence on the nature of the ions; interestingly Gdn.HCl
increased the rate of fibrillation, although much less than NaCl. Different
ions also differentially affected the rate of nucleation and the rate of fibril
elongation.
Conformational Constraints for Amyloid Fibrillation: The Importance of
Being Unfolded.
Biochim
Biophys Acta. (2004) May 6;1698(2):131-53
Uversky, V. N. &
Fink, A. L.,
Recent reports give strong support to the idea that amyloid fibril formation and the subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. In this review, recent findings are surveyed to illustrate that protein fibrillogenesis requires a partially folded conformation. This amyloidogenic conformation is relatively unfolded, and shares many structural properties with the pre-molten globule state, a partially folded intermediate frequently observed in the early stages of protein folding and under some equilibrium conditions. The inherent flexibility of such an intermediate is essential in allowing the conformational rearrangements necessary to form the core cross-beta structure of the amyloid fibril.
The flavonoid baicalein inhibits
fibrillation of alpha-synuclein and disaggregates existing fibrils.
Zhu
M, Rajamani S, Kaylor J, Han S, Zhou F, Fink AL.
The aggregation of alpha-synuclein has been implicated as
a critical step in the development of Parkinson's disease. Parkinson's disease
is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons
from the substantia nigra; currently, no cure exists. Baicalein is a flavonoid
with antioxidant properties; upon oxidation, it forms several products
including quinones. We show here that low micromolar concentrations of
baicalein, and especially its oxidized forms, inhibit the formation of
alpha-synuclein fibrils. In addition, existing fibrils of alpha-synuclein are
disaggregated by baicalein. The product of the inhibition reaction is
predominantly a soluble oligomer of alpha-synuclein, in which the protein
molecules have been covalently modified by baicalein quinone to form a Schiff
base with a lysine side chain in alpha-synuclein. The binding of baicalein was
abolished by conversion of the Tyr residues into Phe, demonstrating that Tyr is
involved in the interaction of alpha-synuclein with baicalein. In
disaggregation baicalein causes fragmentation throughout the length of the
fibril. These observations suggest that baicalein and similar compounds may
have potential as therapeutic leads in combating Parkinson's disease and that
diets rich in flavonoids may be effective in preventing the disorder.
Annular oligomeric amyloid intermediates observed by In-situ AFM
J
Biol Chem. 2004, Jun 4; 279(2): 24452-9
Zhu,M.; Han,S.;
Zhou,F.; Carter,S.A.; Fink,A.L.
Amyloidoses and related protein deposition diseases involve the
transformation of normally soluble proteins into insoluble deposits, usually
fibrillar in nature. Although it was originally assumed that the fibrils were
the toxic species, this assumption has recently been called into question.
Accumulating evidence in several systems suggests that oligomeric intermediates
on the aggregation pathway may be toxic. In the present study we used in-situ
atomic force microscopy to monitor aggregation in aqueous solution in real
time. The sample used was an amyloidogenic immunoglobulin light chain, involved
in AL or light chain amyloidosis. The nature of the observed oligomeric
intermediates was dependent on the conditions of incubation, especially pH and
ionic strength. Several different aggregation intermediates with a variety of
morphologies, including annular or torus-shaped species, were observed. The
data indicate that protein aggregation can be very complex, involving a variety
of different oligomeric intermediates whose population will be determined by
the kinetic and thermodynamic competition between them
Role of Individual Methionines in the Fibrillation of
Methionine-Oxidized alpha-Synuclein
Biochemistry 2004, 43,
4621-4633
Hokenson,M.J.;
Uversky,V.N.; Goers,J.; Yamin,G.; Munishkina,L.A.; Fink,A.L.
The aggregation of normally soluble alpha-synuclein in the dopaminergic
neurons of the substantia nigra is a crucial step in the pathogenesis of
Parkinson's disease. Oxidative stress is believed to be a contributing factor
in this disorder. We have previously established that oxidation of all four
methionine residues in alpha-synuclein (to the sulfoxide, MetO) inhibits
fibrillation of this protein in vitro and that the MetO protein also inhibits
fibrillation of unmodified alpha-synuclein. Here we show that the degree of
inhibition of fibrillation by MetO alpha-synuclein is proportional to the
number of oxidized methionines. This was accomplished be selectively converting
Met residues into Leu, prior to Met oxidation. The results showed that with one
oxidized Met the kinetics of fibrillation were comparable to those for the
control (nonoxidized), and with increasing numbers of methionine sulfoxides the
kinetics of fibrillation became progressively slower. Electron microscope
images showed that the fibril morphology was similar for all species examined,
although fewer fibrils were observed with the oxidized forms. The presence of
zinc was shown to overcome the Met oxidation-induced inhibition. Interestingly,
substitution of Met by Leu led to increased propensity for aggregation (soluble
oligomers) but slower formation of fibrils
Stimulation
of insulin fibrillation by urea-induced intermediates.
J
Biol Chem. 2004 Apr 9;279(15):14999-5013. Epub 2004 Jan 20
Ahmad A, Millett IS, Doniach S, Uversky VN, Fink
AL.
Fibrillar deposits of insulin
cause serious problems in implantable insulin pumps, commercial production of
insulin, and for some diabetics. We performed a systematic investigation of the
effect of urea-induced structural perturbations on the mechanism of
fibrillation of insulin. The addition of as little as 0.5 m urea to zinc-bound
hexameric insulin led to dissociation into dimers. Moderate concentrations of
urea led to accumulation of a partially unfolded dimer state, which dissociates
into an expanded, partially folded monomeric state. Very high concentrations of
urea resulted in an unfolded monomer with some residual structure. The addition
of even very low concentrations of urea resulted in increased fibrillation. Accelerated
fibrillation correlated with population of the partially folded intermediates,
which existed at up to 8 m urea, accounting for the formation of substantial
amounts of fibrils under such conditions. Under monomeric conditions the
addition of low concentrations of urea slowed down the rate of fibrillation,
e.g. 5-fold at 0.75 m urea. The decreased fibrillation of the monomer was due
to an induced non-native conformation with significantly increased
alpha-helical content compared with the native conformation. The data indicate
a close-knit relationship between insulin conformation and propensity to
fibrillate. The correlation between fibrillation and the partially unfolded
monomer indicates that the latter is a critical amyloidogenic intermediate in
insulin fibrillation.
The
role of protein-water interactions and electrostatics in a-synuclein fibril formation.
Biochemistry (2003) 43,
3289-3300
Munishkina, L. A.,
Henriques, J., Uversky. V. N. and Fink, A. L.
Deposition of misfolded alpha-synuclein
is a critical factor in several neurodegenerative disorders. Filamentous
alpha-synuclein is the major component of Lewy bodies and Lewy neurites, the
intracellular inclusions in the dopaminergic neurons of the substantia nigra,
which are considered the pathological hallmark of Parkinson's disease. We show
here that anions induce partial folding of alpha-synuclein at neutral pH,
forming a critical amyloidogenic intermediate, which leads to significant
acceleration of the rate of fibrillation. The magnitude of the accelerating
effect generally followed the position of the anions in the Hofmeister series,
indicating a major role of protein-water-anion interactions in the process at
salt concentrations above 10 mM. Below this concentration, electrostatic
effects dominated in the mechanism of anion-induced fibrillation. The
acceleration of fibrillation by anions was also dependent on the cation.
Moderate concentrations of anions affected both the rates of nucleation and the
elongation of alpha-synuclein fibrillation, primarily via their effect on the
interaction of the protein with water.
FASEB
J
(2004).Jun; 18(9): 962-4.
Li J, Zhu M,
Manning-Bog, A., Di Monte, D., & Fink, A. L.
Protein deposition diseases
involve the aggregation of normally soluble proteins, leading to both fibrillar
and amorphous deposits. The aggregation of alpha-synuclein is associated with
Parkinson's disease, and the aggregation of the Abeta peptide is associated
with Alzheimer's disease. Here we show that L-dopa, dopamine, and other
catecholamines dissolve fibrils of alpha-synuclein and Abeta peptide generated
in vitro. The catecholamines also inhibited the fibrillation of these proteins.
In addition, intraneuronal alpha-synuclein deposits formed in a mouse model
were dissolved by incubation of tissue slices with L-dopa. These catecholamines
are susceptible to oxidative breakdown, and we show that oxidation products are
more effective than the parent compounds in inhibition. The ability to dissolve
fibrils provides a new approach for studying mechanisms and consequences (e.g.,
the relationship between fibril formation and neurodegeneration) of protein
aggregation. It is also likely to help in the development of strategies for the
prevention and treatment of protein deposition diseases. uclein * Abeta peptide
* catecholamines * oxidation
Spectral properties of Thioflavine T and its complexes with amyloid
fibrils.
Journal of Applied Spectroscopy (Moscow) (2003) 70, 765-773.
Voropay E.S., Samtsov M.P., Kaplevsky K.N., Maskevich A.A.,
Stepuro V.I., Povarova O.I., Kuznetsova I.M., Tutoverov K.K., Fink A.L.,
Uversky V.N.
Comparative analysis of the
absorption and fluorescence spectra and fluorescence excitation spectra of
thioflavin T (ThT) in various solvents and in the composition of amyloid
fibrils has shown that ThT, when excited in the region of the long-wavelength
absorption band, fluoresces in the spectral region with a maximum at 478-484
nm. The appearance in aqueous and
alcohol solutions of a fluorescence band with a maximum near 440 nm has been
attributed to the presence in the composition of the ThT preparations of an
impurity with an absorption band in the 340-350-nm range. The literature data
showing that in glycerol ThT has a wide fluorescence spectrum with two maxima
are due to the artifact connected with the use of a high concentration of the
dye. It has been suggested that the cause of the low quantum yield of ThT aqueous
and alcohol solutions is the breakage of the system of conjugated bonds due to
the reorientation of the benzothiozole and benzaminic rings of ThT in the
excited state with respect to one another. The main factor determining the high
quantum yield of fluorescence of ThT incorporated in fibrils is the steric
restriction of the rotation of the rings about one another under these
conditions. The suggestions made have been verified by the quantum-chemical
calculation of ThT molecule geometry in the ground and excited states.
Partially folded intermediates in insulin
fibrillation.
Biochemistry. 2003 Oct 7;42(39):11404-16
Ahmad A,
Millett IS, Doniach S, Uversky VN, Fink AL.
Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.
A model for amyloid fibril formation in
immunoglobulin light chains based on comparison of amyloidogenic and benign
proteins and specific antibody binding.
Amyloid. 2003 Jun;10(2):97-109
Khurana R, Souillac PO, Coats AC, Minert L, Ionescu-Zanetti C, Carter SA,
Solomon A, Fink AL.
In an attempt to understand the mechanism of amyloid fibril formation in light chain amyloidosis, the properties of amyloidogenic (SMA) and benign (LEN) immunoglobulin light chain variable domains (VL) were compared. The conformations of LEN and SMA were measured using secondary and tertiary structural probes over the pH range from 2 and 8. At all pH values, LEN was more stable than SMA. The CD spectra of LEN at pH 2 were comparable to those of SMA at pH 7.5, indicating that the low pH conformation of LEN closely resembles that of SMA at physiological pH. At low pH, a relatively unfolded intermediate conformation is populated for SMA and rapidly leads to amyloid fibrils. The lack of such an intermediate with LEN, is attributed to sequence differences and accounts for the lack of LEN fibrils in the absence of agitation. A kappa IV-specific monoclonal antibody that recognizes the N-terminal of SMA caused unraveling of the fibrils to the protofilaments and was observed to bind to one end of SMA protofilaments by atomic force microscopy. The antibody result indicates that each protofilament is asymmetric with different ends. A model for the formation of fibrils by SMA is proposed.
The association of alpha-synuclein with
membranes affects bilayer structure, stability, and fibril formation.
J Biol Chem. 2003 Oct
10;278(41):40186-97. Epub 2003 Jul 28.
Zhu M, Li
J, Fink AL.
The aggregation of alpha-synuclein is believed to be a critical factor in the etiology of Parkinson's disease. alpha-Synuclein is an abundant neuronal protein of unknown function, which is enriched in the presynaptic terminals of neurons. Although alpha-synuclein is found predominantly in the cytosolic fractions, membrane-bound alpha-synuclein has been suggested to play an important role in fibril formation. The effects of alpha-synuclein on lipid bilayers of different compositions were determined using fluorescent environment-specific probes located at various depths. alpha-Synuclein-membrane interactions were found to affect both protein and membrane properties. Our results indicate that in addition to electrostatic interactions, hydrophobic interactions are important in the association of the protein with the bilayer, and lead to disruption of the membrane. The latter was observed by atomic force microscopy and fluorescent dye leakage from vesicles. The kinetics of alpha-synuclein fibril formation were significantly affected by the protein association and subsequent membrane disruption, and reflected the conformation of alpha-synuclein. The ability of alpha-synuclein to disrupt membranes correlated with the binding affinity of alpha-synuclein for the particular membrane composition, and to the induced helical conformation of alpha-synuclein. Protofibrillar or fibrillar alpha-synuclein caused a much more rapid destruction of the membrane than soluble monomeric alpha-synuclein, indicating that protofibrils (oligomers) or fibrils are likely to be significantly neurotoxic.
A general model for amyloid fibril
assembly based on morphological studies using atomic force microscopy.
Biophys J. 2003 Aug;85(2):1135-44
Khurana
R, Ionescu-Zanetti C, Pope M, Li J, Nielson L, Ramirez-Alvarado M, Regan L,
Fink AL, Carter SA.
Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.
Nuclear localization of alpha-synuclein
and its interaction with histones.
Biochemistry. 2003 Jul 22;42(28):8465-71
Goers J,
Manning-Bog AB, McCormack AL, Millett IS, Doniach S, Di Monte DA, Uversky VN,
Fink AL.
The aggregation of alpha-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of alpha-synuclein under physiologic and pathological conditions is unknown, and the mechanism of alpha-synuclein aggregation is not well understood. Here we show that alpha-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of alpha-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of alpha-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying alpha-synuclein pathophysiology.
Tau filaments from human brain and from
in vitro assembly of recombinant protein show cross-beta structure.
Proc Natl Acad Sci U S A. 2003 Jul
22;100(15):9034-8. Epub 2003 Jul 09
Berriman
J, Serpell LC, Oberg KA, Fink AL, Goedert M, Crowther RA.
Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 2QH, United Kingdom.
Abnormal filaments consisting of hyperphosphorylated microtubule-associated protein tau form in the brains of patients with Alzheimer's disease, Down's syndrome, and various dementing tauopathies. In Alzheimer's disease and Down's syndrome, the filaments have two characteristic morphologies referred to as paired helical and straight filaments, whereas in tauopathies, there is a wider range of morphologies. There has been controversy in the literature concerning the internal molecular fine structure of these filaments, with arguments for and against the cross-beta structure demonstrated in many other amyloid fibers. The difficulty is to produce from brain pure preparations of filaments for analysis. One approach to avoid the need for a pure preparation is to use selected area electron diffraction from small groups of filaments of defined morphology. Alternatively, it is possible to assemble filaments in vitro from expressed tau protein to produce a homogeneous specimen suitable for analysis by electron diffraction, x-ray diffraction, and Fourier transform infrared spectroscopy. Using both these approaches, we show here that native filaments from brain and filaments assembled in vitro from expressed tau protein have a clear cross-beta structure.
Structural transformations of oligomeric
intermediates in the fibrillation of the immunoglobulin light chain LEN.
Biochemistry. 2003 Jul 8;42(26):8094-104
Souillac
PO, Uversky VN, Fink AL.
LEN is a kappaIV immunoglobulin light chain variable domain from a patient suffering from multiple myeloma but with no evidence of amyloid fibrils. However, fibrils are formed when LEN solutions are agitated under mildly destabilizing conditions. Surprisingly, an inverse concentration dependence was observed on the kinetics of fibril formation because of the formation of off-pathway soluble oligomers at high protein concentration. Despite the fact that most of the protein is present in the off-pathway intermediates at relatively early times of aggregation, eventually all the protein forms fibrils. Thus, a structural rearrangement from the non fibril-prone off-pathway oligomers to a more fibril-prone species must occur. A variety of techniques were used to monitor changes in the size, secondary structure, solvent accessibility, and intrinsic stability of the oligomers, as a function of incubation time. The structural rearrangement was accompanied by a significant increase of disordered secondary structure, an increase in solvent accessibility, and a decrease in intrinsic stability of the soluble oligomeric species. We conclude that fibrils arise from the oligomers containing a less stable conformation of LEN, either directly or via dissociation. This is the first fibrillating system in which soluble off-pathway oligomeric intermediates have been shown to be the major transient species and in which fibrillation occurs from a relatively unfolded conformation present in these intermediates.
Certain metals trigger fibrillation of
methionine-oxidized alpha-synuclein.
J Biol Chem. 2003 Jul 25;278(30):27630-5.
Epub 2003 May 16
Yamin G,
Glaser CB, Uversky VN, Fink AL.
The aggregation and fibrillation of alpha-synuclein has been implicated as a key step in the etiology of Parkinson's disease and several other neurodegenerative disorders. In addition, oxidative stress and certain environmental factors, including metals, are believed to play an important role in Parkinson's disease. Previously, we have shown that methionine-oxidized human alpha-synuclein does not fibrillate and also inhibits fibrillation of unmodified alpha-synuclein (Uversky, V. N., Yamin, G., Souillac, P. O., Goers, J., Glaser, C. B., and Fink, A. L. (2002) FEBS Lett. 517, 239-244). Using dynamic light scattering, we show that the inhibition results from stabilization of the monomeric form of Met-oxidized alpha-synuclein. We have now examined the effect of several metals on the structural properties of methionine-oxidized human alpha-synuclein and its propensity to fibrillate. The presence of metals induced partial folding of both oxidized and non-oxidized alpha-synucleins, which are intrinsically unstructured under conditions of neutral pH. Although the fibrillation of alpha-synuclein was completely inhibited by methionine oxidation, the presence of certain metals (Ti3+, Zn2+, Al3+, and Pb2+) overcame this inhibition. These findings indicate that a combination of oxidative stress and environmental metal pollution could play an important role in triggering the fibrillation of alpha-synuclein and thus possibly Parkinson's disease.
Nitration inhibits fibrillation of human
alpha-synuclein in vitro by formation of soluble oligomers.
FEBS Lett. 2003 May 8;542(1-3):147-52
Yamin G,
Uversky VN, Fink AL.
The aggregation of alpha-synuclein in dopaminergic neurons is a critical factor in the etiology of Parkinson's disease (PD). Oxidative and nitrative stress is also implicated in PD. We examined the effect of nitration on the propensity of alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited by nitration, due to the formation of stable soluble oligomers (apparently octamers). More importantly the presence of sub-stoichiometric concentrations of nitrated alpha-synuclein led to inhibition of fibrillation of non-modified alpha-synuclein. These observations suggest that nitration of soluble alpha-synuclein may be a protective factor in PD, rather than a causative one.
Prediction of the association state of
insulin using spectral parameters.
J Pharm Sci. 2003 Apr;92(4):847-58
Uversky
VN, Garriques LN, Millett IS, Frokjaer S, Brange J, Doniach S, Fink AL.
Institute for Biological
Instrumentation, Russian Academy of Sciences, 142292 Pushchino, Moscow Region,
Russia. uversky@hydrogen.ucsc.edu
Human insulin exists in different association states, from monomer to hexamer, depending on the conditions. In the presence of zinc the "normal" state is a hexamer. The structural properties of 20 variants of human insulin were studied by near-UV circular dichroism, fluorescence spectroscopy, and small-angle X-ray scattering (SAXS). The mutants showed different degrees of association (monomer, dimers, tetramers, and hexamers) at neutral pH. A correlation was shown between the accessibility of tyrosines to acrylamide quenching and the degree of association of the insulin mutants. The near-UV CD spectra of the insulins were affected by protein association and by mutation-induced structural perturbations. However, the shape and intensity of difference CD spectra, obtained by subtraction of the spectra measured in 20% acetic acid (where all insulin species were monomeric) from the corresponding spectra measured at neutral pH, correlate well with the degree of insulin association. In fact, the near-UV CD difference spectra for monomeric, dimeric, tetrameric, and hexameric insulin are very distinctive, both in terms of intensity and shape. The results show that the spectral properties of the insulins reflect their state of association, and can be used to predict their oligomeric state. Copyright 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:847-858, 2003
Polycation-induced oligomerization and
accelerated fibrillation of human alpha-synuclein in vitro.
Protein Sci. 2003 Apr;12(4):702-7
Goers J,
Uversky VN, Fink AL.
The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in Parkinson's disease and several other neurodegenerative disorders known as synucleinopathies. The effect of different factors on the process of fibril formation has been intensively studied in vitro. We show here that alpha-synuclein interacts with different unstructured polycations (spermine, polylysine, polyarginine, and polyethyleneimine) to form specific complexes. In addition, the polycations catalyze alpha-synuclein oligomerization. The formation of alpha-synuclein-polycation complexes was not accompanied by significant structural changes in alpha-synuclein. However, alpha-synuclein fibrillation was dramatically accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the nature of the polymer, its length, and concentration. The results illustrate the potential critical role of electrostatic interactions in protein aggregation, and the potential role of naturally occurring polycations in modulating alpha-synuclein aggregation.
Biophysical
properties of a-synuclein and its role in
Parkinson’s disease.
In
Recent Res. Dev. Proteins, 1
(2002), 153-186.
Uversky, V. N. and Fink, A. L.
Parkinson’s disease (PD) is the second most common neurodegenerative disease, and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of α-synuclein in the form of intracellular proteinaceous aggregates (Lewy bodies and Lewy neuritis) has been implicated as a causative factor in this disease, as well as in several other neurodegenerative disorders, including dementia with Lewy bodies Lewy body variant of Alzheimer’s disease, multiple system atrophy and Hallervorden-Spatz disease. Thus, the aggregated forms of α-synuclein play a crucial role in the pathogenesis of the synucleinopathies. However, the molecular mechanisms underlying α-synuclein aggregation into specific filamentous inclusions remained unknown until recently. An intriguing aspect of this problem was that α-synuclein belongs to the class of natively unfolded proteins characterized by little or no ordered structure under physiological conditions. This raised the question of how an essentially disordered protein could be transformed into highly organized fibrils. The first part of this review is devoted to the search for an answer to this question and deals with the description of structural properties of α-synuclein and conformational behavior of this protein under variety of experimental conditions, as well as with the analysis of the effects of these same conditions on fibrillation kinetics of human recombinant α-synuclein.
Two different missense mutations in the α-synuclein gene, corresponding to A30P and A53TL substitutions in the protein, have been identified in a small number of kindreds from human protein at seven positions, including position 53, where the murine protein contains a threonine residue, however neither Parkinsonian symptoms nor Lewy bodies have been observed in aged mice. Besides α-synuclein, the family of human synucleins includes β- and γ-synucleins, with all three proteins showing large sequence homology. Structural consequences of amino substitutions on α-synuclein structure, conformational behavior and aggregation are considered in the second part of review.
The etiology of PD
is unknown, but recent work has shown that, except in rare cases, there appears
to be no direct genetic basis. However, a positive correlation between the
prevalence of PD and industrialization has been recognized. Particularly,
several epidemiological studies have implicated such environmental factors as
pesticides, herbicides and heavy metals in the PD origin and this disorder is
now considered likely to be an “environmental” disease. More specifically,
occupational exposure to specific metals, especially manganese, copper, lead,
iron, mercury, zinc, and aluminum, was shown to be a risk factor for PD.
Moreover, elevated levels of several of these metals have also been reported in
the substantia nigra of PD subjects. Furthermore, farming, rural living,
well-water drinking and exposure to agricultural chemicals are all conditions
that have been associated with an increased risk for PD. The third part of the
review summarizes data showing that the environmental factors can directly
affect the aggregation of α-synuclein,
providing a critical link between toxicant exposures and the pathogenesis of α-synuclein-containing inclusions in
idiopathic PD.
Structural and functional properties of
Yersinia pestis Caf1 capsular antigen and their possible role in fulminant
development of primary pneumonic plague.
J Proteome Res. 2002 Jul-Aug;1(4):307-15
Abramov
VM, Vasiliev AM, Khlebnikov VS, Vasilenko RN, Kulikova NL, Kosarev IV,
Ishchenko AT, Gillespie JR, Millett IS, Fink AL, Uversky VN.
Institute of Immunological
Engineering, 142380 Lyubuchany, Moscow Region, Russia.
Yersinia pestis capsular antigen Caf1 is shown to be a beta-structural protein that in polymeric form possesses very high conformational stability. Different approaches show that a dimer is the minimal cooperative block of Caf1 adhesin. Caf1 dimer interacts effectively with IL-1 receptors of human macrophage and epithelial cells. The specificity of such interaction is confirmed by the inhibition of IL-1alpha binding by Caf1. The Caf1 role in pneumonic plague pathogenesis is discussed.
Effect of zinc and temperature on the
conformation of the gamma subunit of retinal phosphodiesterase: a natively
unfolded protein.
J Proteome Res. 2002 Mar-Apr;1(2):149-59
Uversky
VN, Permyakov SE, Zagranichny VE, Rodionov IL, Fink AL, Cherskaya AM, Wasserman
LA, Permyakov EA.
Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino, Russia. uversky@hydrogen.ucsc.edu
The cyclic GMP phosphodiesterase gamma-subunit (PDEgamma) was shown to belong to the family of natively unfolded proteins. Increasing temperature transforms the protein into a more ordered (but still relatively disordered) conformation. The C-terminal part of PDEgamma has a high-affinity zinc-binding site (Kd approximately 1 microM), with His75 and His79 being directly involved into the coordination of Zn2+. Zinc-loaded protein remains effectively unfolded. Possible implications of these findings to the functioning of PDEgamma are discussed.
Lipid binding inhibits alpha-synuclein
fibril formation.
J Biol Chem. 2003 May 9;278(19):16873-7.
Epub 2003 Mar 05
Zhu M,
Fink AL.
Parkinson's disease is the second most common neurodegenerative disorder, and the cause is unknown; however, substantial evidence implicates the aggregation of alpha-synuclein as a critical factor in the etiology of the disease. alpha-Synuclein is a relatively abundant brain protein of unknown function, and the purified protein is intrinsically unfolded. The amino acid sequence has seven repeats with an apolipoprotein lipid-binding motif, which are predicted to form amphiphilic helices. We have investigated the interaction of alpha-synuclein with lipid vesicles of different sizes and properties by monitoring the effects on the conformation of the protein and the kinetics of fibrillation. The nature of the interaction of alpha-synuclein with vesicles was highly dependent on the phospholipid composition, the ratio of alpha-synuclein to phospholipid, and the size of the vesicles. The strongest interactions were between alpha-synuclein and vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphate/1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho-RAC-(1-glycerol)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine and involved formation of helical structure in alpha-synuclein. A strong correlation was observed between the induction of alpha-helix in alpha-synuclein and the inhibition of fibril formation. Thus, helical, membrane-bound alpha-synuclein is unlikely to contribute to aggregation and fibrillation. Given that a significant fraction of alpha-synuclein is membrane-bound in dopaminergic neurons, this observation has significant physiological significance.
Conformational behavior and aggregation
of alpha-synuclein in organic solvents: modeling the effects of membranes.
Biochemistry. 2003 Mar 11;42(9):2720-30
Munishkina
LA, Phelan C, Uversky VN, Fink AL.
Intracellular proteinaceous inclusions (Lewy bodies and Lewy neurites) of alpha-synuclein are pathological hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies (DLB), and multiple systemic atrophy. The molecular mechanisms underlying the aggregation of alpha-synuclein into such filamentous inclusions remain unknown, although many factors have been implicated, including interactions with lipid membranes. To model the effects of membrane fields on alpha-synuclein, we analyzed the structural and fibrillation properties of this protein in mixtures of water with simple and fluorinated alcohols. All solvents that were studied induced folding of alpha-synuclein, with the common first stage being formation of a partially folded intermediate with an enhanced propensity to fibrillate. Protein fibrillation was completely inhibited due to formation of beta-structure-enriched oligomers with high concentrations of methanol, ethanol, and propanol and moderate concentrations of trifluoroethanol (TFE), or because of the appearance of a highly alpha-helical conformation at high TFE and hexafluoro-2-propanol concentrations. At least to some extent, these conformational effects mimic those observed in the presence of phospholipid vesicles, and can explain some of the observed effects of membranes on alpha-synuclein fibrillation.
Disorder in the nuclear pore complex: the
FG repeat regions of nucleoporins are natively unfolded.
Proc Natl Acad Sci U S A. 2003 Mar
4;100(5):2450-5. Epub 2003 Feb 25
Denning
DP, Patel SS, Uversky V, Fink AL, Rexach M.
Department of Biological Sciences,
Stanford University, Stanford, CA 94305, USA.
Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of "natively unfolded" proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds
Conformational behavior of human alpha-synuclein
is modulated by familial Parkinson's disease point mutations A30P and A53T.
Neurotoxicology. 2002 Oct;23(4-5):553-67
Li J,
Uversky VN, Fink AL.
Structural properties and response to changes in the environment of wild-type (WT), A30P and A53T alpha-synucleins, as well as their propensity to aggregate orform fibrils, were compared by a variety of biophysical methods, including far-UV CD, FTIR, SAXS, static light scattering and Thioflavin T (TFT) fluorescence. All three proteins were natively unfolded under physiological conditions but adopted identical partially-folded conformations under conditions of acidic pH or high temperature. The initial kinetic event in the fibrillation of all three alpha-synucleins was shown to be the formation of a partially-folded intermediate with properties close to those described for these proteins at acidic pH or at high temperatures. Both mutants showed a greater propensity to form non-fibrillar aggregates than wild-type protein. All three proteins formed fibrils faster in the presence of heparin, although substantially higher concentrations were required for the A30P mutant. In contrast to the wild-type and A53T proteins, in which fibrillation was further accelerated by the presence of the pesticide diethyldithiocarbamate (DDC), the A30P mutant was inhibited by DDC. The mutant proteins had significantly lower affinity for DDC than the WT. A model of the effect of mutations on the aggregation behavior of alpha-synuclein is proposed, which explains the different effects of exogenous agents on the three proteins, based on different kinetic partitioning along pathways leading to fibrils and to non-fibrillar aggregates.
Synergistic effects of pesticides and
metals on the fibrillation of a-synuclein: Implications for Parkinson’s disease
Neurotoxicology (2002) 23(4-5), 527-536
Aggregation of a-synuclein has been implicated in the formation of proteinaceous inclusions in the brain (Lewy bodies, Lewy neurites) that are characteristic of neurodegenerative diseases such as Parkinson’s disease and dementia with Lewy bodies. The etiology of Parkinson’s disease is unknown, but recent work has shown that, except in rare cases, there appears to be no direct genetic basis. However, several studies have implicated environmental factors, especially pesticides and metals. Here we show that certain pesticides and metals induce a conformational change in a-synuclein and directly accelerate the rate of formation of a-synuclein fibrils in vitro. In addition, the simultaneous presence of metal and pesticide led to synergistic effects on the rate of fibrillation. We propose a model in which environmental factors, in conjunction with genetic susceptibility, may form the underlying molecular basis for idiopathic Parkinson’s disease.
Conformational prerequisites for
alpha-lactalbumin fibrillation.
Biochemistry. 2002 Oct 15;41(41):12546-51
Goers J,
Permyakov SE, Permyakov EA, Uversky VN, Fink AL.
Bovine alpha-lactalbumin, a small acidic Ca(2+)-binding milk protein, formed amyloid fibrils at low pH, where it adopted the classical molten globule-like conformation. Fibrillation was accompanied by a dramatic increase in the beta-structure content and a characteristic increase in the thioflavin T fluorescence intensity. S-(Carboxymethyl)-alpha-lactalbumin, a disordered form of the protein with three out of four disulfide bridges reduced, was even more susceptible to fibrillation. Other partially folded conformations induced in the intact protein at neutral pH, either by the removal of Ca(2+) or by the binding of Zn(2+) to the Ca(2+)-protein complex, did not fibrillate, although Zn(2+)-loaded alpha-lactalbumin precipitated out of solution as amorphous aggregates. Our data suggest that the transformation of a protein into an essentially unfolded (thus, highly flexible) conformation is required for successful fibril formation, whereas more rigid (but still flexible) species may form amorphous aggregates.
Surface-catalyzed amyloid fibril formation.
J Biol Chem. 2002 Dec
27;277(52):50914-22. Epub 2002 Sep 27
Zhu M,
Souillac PO, Ionescu-Zanetti C, Carter SA, Fink AL.
Department of Chemistry and
Biochemistry and the Department of Physics, University of California, Santa
Cruz, California 95064, USA.
Light chain (or AL) amyloidosis is characterized by the pathological deposition of insoluble fibrils of immunoglobulin light chain fragments in various tissues, walls of blood vessels, and basement membranes. In the present investigation, the in vitro assembly of a recombinant amyloidogenic light chain variable domain, SMA, on various surfaces was monitored using atomic force microscopy. SMA formed fibrils on native mica at pH 5.0, conditions under which predominantly amorphous aggregates form in solution. Fibril formation was accelerated significantly on surfaces compared with solution; for example, fibrils grew on surfaces at significantly faster rates and at much lower concentrations than in solution. No fibrils were observed on hydrophobic or positively charged surfaces or at pH >7.0. Two novel types of fibril growth were observed on the surface: bidirectional linear assembly of oligomeric units, and linear growth from preformed amorphous cores. In addition to catalyzing the rate of fibrillation, the mechanism of fibril formation on the surfaces was significantly different from in solution, but it may be more physiologically relevant because in vivo the deposits are associated with surfaces.
Amino acid determinants of
alpha-synuclein aggregation: putting together pieces of the puzzle.
FEBS Lett. 2002 Jul 3;522(1-3):9-13
Uversky
VN, Fink AL.
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA. uversky@hydrogen.ucsc.edu
Parkinson's disease is the second most common neurodegenerative disease, and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of alpha-synuclein in the form of intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) have been implicated as a causative factor in this disease, as well as in several other neurodegenerative disorders, including dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, multiple system atrophy and Hallervorden-Spatz disease. Thus, the aggregated forms of alpha-synuclein play a crucial role in the pathogenesis of the synucleinopathies. However, the molecular mechanisms underlying alpha-synuclein aggregation into specific filamentous inclusions remained unknown until recently. Data on the aggregation and fibrillation properties of human alpha-, beta- and gamma-synucleins, mouse alpha-synuclein and familial Parkinson's disease mutants of human alpha-synuclein (A30P and A53T) are analyzed in order to shed light on the amino acid determinants of synuclein aggregation.
The Saccharomyces cerevisiae nucleoporin
Nup2p is a natively unfolded protein.
J Biol Chem. 2002 Sep 6;277(36):33447-55.
Epub 2002 Jun 13
Denning
DP, Uversky V, Patel SS, Fink AL, Rexach M.
Department of Biological Sciences, Stanford University, Stanford, California 94305, USA.
Little is known about the structure of the individual nucleoporins that form eukaryotic nuclear pore complexes (NPCs). We report here in vitro physical and structural characterizations of a full-length nucleoporin, the Saccharomyces cerevisiae protein Nup2p. Analyses of the Nup2p structure by far-UV circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, protease sensitivity, gel filtration, and sedimentation velocity experiments indicate that Nup2p is a "natively unfolded protein," belonging to a class of proteins that exhibit little secondary structure, high flexibility, and low compactness. Nup2p possesses a very large Stokes radius (79 A) in gel filtration columns, sediments slowly in sucrose gradients as a 2.9 S particle, and is highly sensitive to proteolytic digestion by proteinase K; these characteristics suggest a structure of low compactness and high flexibility. Spectral analyses (CD and FTIR spectroscopy) provide additional evidence that Nup2p contains extensive regions of structural disorder with comparatively small contributions of ordered secondary structure. We address the possible significance of natively unfolded nucleoporins in the mechanics of nucleocytoplasmic trafficking across NPCs.
Methionine oxidation inhibits fibrillation of human a-synuclein in vitro
FEBS Letters 517 (2002) 239-244
Vladimir N. Uversky, Ghiam Yamin, Pierre O. Souillac, John
Goers, Charles B. Glaser and Anthony L. Fink
We examined the effect of methionine oxidation of human recombinant a-synuclein on its structural properties and propensity to fibrillate. Both oxidized and non-oxidized a-synucleins were natively unfolded under conditions of neutral pH, with the oxidized protein being slightly more disordered. Both proteins adopted identical partially folded conformations under conditions of acidic pH. The fibrillation of a-synuclein at neutral pH was completely inhibited by methionine oxidation. This inhibitory effect was eliminated at low pH. The addition of oxidized a-synuclein to the unoxidized form led to a substantial inhibition of a-synuclein fibrillation.
Unraveling multistate unfolding of rabbit muscle creatine
kinase.
Biochim Biophys Acta. 2002 Apr 1;1596(1):138-55.
Kuznetsova IM, Stepanenko
OV, Turoverov KK, Zhu L, Zhou JM, Fink AL, Uversky VN.
Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia.
GdmCl-induced unfolding of rabbit muscle creatine kinase, CK, has been studied by a variety of physico-chemical methods including near and far UV CD, SEC, intrinsic fluorescence (intensity, anisotropy and lifetime) as well as intensity and lifetime of bound ANS fluorescence. The formation of several stable unfolding intermediates, some of which were not observed previously, has been established. This was further confirmed by representation of fluorescence data in terms of "phase diagram", i.e. I(lambda1) versus I(lambda2) dependence, where I(lambda1) and I(lambda2) are fluorescence intensity values measured on wavelengths lambda(1) and lambda(2) under the different experimental conditions for a protein undergoing structural transformations. The unfolding behavior of CK was shown to be strongly affected by association of partially folded intermediates. A model of CK unfolding, which takes into account both structural perturbations and association of partially folded intermediates has been elaborated.
Accelerated a-Synuclein
Fibrillation in Crowded Milieu
FEBS Letters 515 (2001) 99-103
Vladimir
N. Uversky, Elisa M. Cooper, Kiowa S. Bower,
Jie Li and Anthony L. Fink
Parkinson’s disease is the second most common age-related neurodegenerative disease, resulting from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of a-synuclein has been implicated as a causative factor in the disease, and the process of fibril formation has been intensively studied in vitro with dilute protein solutions. However, the intracellular environment of proteins is crowded with other macromolecules, whose concentration can reach 400 g/L. To address this discrepancy, the effect of molecular crowding on a-synuclein fibrillation has being studied. The addition of high concentrations of different polymers (proteins, polysaccharides and polyethyleneglycols) dramatically accelerated a-synuclein fibrillation in vitro. The magnitude of the accelerating effect depended on the nature of the polymer, its length and concentration. Our results suggest that the major factor responsible for the accelerated fibrillation under crowded conditions is the excluded volume.
The chicken-egg
scenario of protein folding revisited.
FEBS Lett. 2002
Mar 27;515(1-3):79-83
Uversky VN, Fink AL.
Institute
for Biological Instrumentation, Russian Academy of Sciences, 142292, Moscow
Region, Pushchino, Russia. uversky@hydrogen.ucsc.edu
What is the first step in protein folding - hydrophobic collapse (compaction) or secondary structure formation? It is still not clear if the major driving force in protein folding is hydrogen bonding or hydrophobic interactions or both. We analyzed data on the conformational characteristics of 41 globular proteins in native and partially folded conformational states. Our analysis shows that a good correlation exists between relative decrease in hydrodynamic volume and increase in secondary structure content. No compact equilibrium intermediates lacking secondary structure, or highly ordered non-compact species, were found. This correlation provides experimental support for the hypothesis that hydrophobic collapse occurs simultaneously with formation of secondary structure in the early stages of the protein folding.
Effect of association state and conformational stability on the
kinetics of immunoglobulin light chain amyloid fibril formation at
physiological pH
J Biol Chem (2002) 277:15, 12657-12665
Pierre O. Souillac, Vladimir
N. Uversky, Ian S. Millett, Ritu Khurana, Sebastian Doniach, and Anthony L.
Fink
Light chain amyloidosis involves the systemic deposition of fibrils in patients overproducing monoclonal immunoglobulin light chains. The kinetics of fibril formation of LEN, a benign light chain variable domain, were investigated at physiological pH in the presence of urea. Despite the lack of in vivo fibril formation, LEN readily forms fibrils in vitro under mildly destabilizing conditions. The effect of low to moderate concentrations of urea on the conformation, association state, stability and kinetics of fibrillation of LEN were investigated. The conformation of LEN was only slightly affected by the addition of up to 4 M urea. The fibrillation kinetics were highly dependent on protein and urea concentrations, becoming faster with decreasing protein concentration and increasing urea concentration. Changes in spectral probes were concomitant to fibril formation throughout the protein and urea concentration ranges, indicating the absence of off-pathway oligomeric species or amorphous aggregates prior to fibril formation. Reducing the amount of dimers initially present in solution by either decreasing the protein concentration or adding urea resulted in faster fibril formation. Thus, increasing concentrations of urea, by triggering dissociation of dimeric LEN, lead to increased rates of fibrillation.
Elucidation of the molecular mechanism during the early events in
immunoglobulin light chain amyloid fibrillation: Evidence for an off-pathway
oligomer at acidic pH
J Biol Chem (2002) 277:15, 12666-12679
Pierre O. Souillac, Vladimir
N. Uversky, Ian S. Millett, Ritu Khurana, Sebastian Doniach, and Anthony L.
Fink
Light chain amyloidosis (AL amyloidosis) involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle X-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway, and leads to formation of the off-pathway intermediate.
Biophysical Properties of the Synucleins and Their Propensities to
Fibrillate: Inhibition of a-Synuclein Assembly by b- and g-Synucleins
J Biol Chem (2002) 277:14, 11970-11978
Vladimir N. Uversky, Jie Li, Pierre
Souillac, Ross Jakes, Michel Goedert and Anthony L. Fink
The pathological hallmark of Parkinson’s disease is the presence of intracellular inclusions, Lewy bodies and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous a-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson’s disease and several other neurodegenerative diseases. Two homologous proteins, b- and g-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. b-Synuclein, which lacks 11 central hydrophobic residues compared to its homologs, exhibited the properties of a random coil, whereas a- and g-synucleins were slightly more compact and structured. g-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to a-synuclein, b-and g-synucleins inhibit a-synuclein fibril formation. Complete inhibition of a-synuclein fibrillation was observed at 4:1 molar excess of b-and g-synucleins. No significant incorporation of b-synuclein into the fibrils was detected. The lack of fibrils formed by b-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed.
Heparin and Other Glycosaminoglycans
Stimulate the Formation of Amyloid Fibrils from a-Synuclein in vitro
Biochemistry (2002) 41:5, 1502-1511
Jeffrey A. Cohlberg, Jie Li, Vladimir N. Uversky and Anthony L. Fink
Parkinson’s disease is the second most common neurodegenerative disease and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of a-synuclein has been implicated as a causative factor in the disease. Glycosaminoglycans (GAGs) are routinely found associated with amyloid deposits in most amyloidosis diseases, and there is evidence to support an active role of GAGs in amyloid fibril formation in some cases. In contrast to the extracellular amyloid deposits, the a-synuclein deposits in Lewy body diseases are intracellular, and thus it is less clear whether GAGs may be involved. In order to determine whether the presence of GAGs does affect the fibrillation of a-synuclein, the kinetics of fibril formation were investigated in the presence of a number of GAGs and other charged polymers. Certain GAGs (heparin, heparan sulfate) and other highly sulfated polymers (dextran sulfate) were found to significantly stimulate the formation of a-synuclein fibrils. Interestingly, the interaction of GAGs with a-synuclein is quite specific, since some GAGs, e. g. keratan sulfate, had negligible effect. Heparin not only increased the rate of fibrillation, but also apparently increased the yield of fibrils. The molar ratio of heparin to a-synuclein, and the incorporation of fluorescein-labeled heparin into the fibrils, demonstrates that the heparin is integrated into the fibrils, and is not just a catalyst for fibrillation. The apparent dissociation constant for heparin in stimulating a-synuclein fibrillation was 0.19 mM, indicating a strong affinity. Similar effects of heparin were observed with the A53T and A30P mutants of a-synuclein. Since there is some evidence that Lewy bodies may contain GAGs, these observations may be very relevant in the context of the etiology of Parkinson’s disease.
The Herbicide Paraquat Causes Up-regulation
and Aggregation of aSynuclein
in Mice
J Biol Chem (2002) 277:3, 1641-1644
Amy B.
Manning-Bog, Alison L. McCormack, Jie Li, Vladimir N. Uversky, Anthony L. Fink,
and Donato A. Di Monte
aSynuclein containing aggregates represent a feature of a variety of neurodegenerative disorders, including Parkinson's disease (PD). However, mechanisms that promote intraneuronal asynuclein assembly remain poorly understood. Because pesticides, particularly the herbicide paraquat, have been suggested to play a role as PD risk factors, the hypothesis that interactions between asynuclein and these environmental agents may contribute to aggregate formation was tested in this study. Paraquat markedly accelerated the in vitro rate of asynuclein fibril formation in a dose-dependent fashion. When mice were exposed to the herbicide, brain levels of asynuclein were significantly increased. This upregulation followed a consistent pattern, with higher asynuclein at 2 days after each of 3 weekly paraquat injections and with protein levels returning to control values by day 7 posttreatment. Paraquat exposure was also accompanied by aggregate formation. Thioflavine S positive structures accumulated within neurons of the substantia nigra pars compacta, and dual labeling and confocal imaging confirmed that these aggregates contained asynuclein. The results suggest that upregulation of asynuclein as a consequence of toxicant insult and direct interactions between the protein and environmental agents are potential mechanisms leading to asynuclein pathology in neurodegenerative disorders.
Trimethylamine-N-Oxide-Induced Folding of a-Synuclein
FEBS Letters (2001) 509:1, 31-35
Vladimir N. Uversky, Jie Li and Anthony L. Fink
The effect of the natural osmolyte trimethylamine-N-oxide (TMAO) on the structural properties and fibril formation of the natively unfolded protein human a-synuclein was studied using several physico-chemical methods. TMAO induced folding of a-synuclein: at moderate concentrations a partially-folded intermediate with enhanced propensity for fibrillation accumulated, at higher concentrations a-synuclein was tightly folded and underwent self-association to form oligomers. The latter conformation was significantly helical and probably represents the physiologically folded form of the protein.
Stabilization of partially folded conformation during alpha-synuclein oligomerization in both purified and cytosolic preparations.
J
Biol Chem. 2001 Nov 23;276(47):43495-8
Uversky VN, Lee HJ, Li J, Fink AL, Lee SJ.
Aggregation of alpha-synuclein is tightly associated with many neurodegenerative diseases, such as Parkinson's disease, dementia with Lewy body, Lewy body variant of Alzheimer's disease, multiple system atrophy, and Hallervorden-Spatz disease, implicating a crucial role of aggregated forms of alpha-synuclein in the pathogenesis. Here, we examined the effect of elevated temperature on the oligomerization and structural changes of alpha-synuclein in the early stage of aggregation and show that self-assembly is crucial for the stabilization of a partially folded conformation. The efficiency of alpha-synuclein oligomerization increased proportional to the temperature increase, both in purified form and in crude cytosolic preparation. This oligomerization coincided with a small but reproducible change in the circular dichroism spectrum and an increase in the 1-anilinonaphthalene-8-sulfonic acid binding. The hydrodynamic dimensions of the dimer measured by size exclusion chromatography suggest a pre-molten globule-like structure. These data suggest that partially folded alpha-synuclein, which is unstable in the monomeric form, is stabilized by self-assembly and that these oligomers may evolve into the fibril nucleus.
Effect of Familial Parkinson’s Disease Point Mutations A30P and
A53T on the Structural Properties, Aggregation and Fibrillation of Human
alpha-Synuclein
Biochemistry (2001) 40:38, 11604-11613
Jie Li,
Vladimir N. Uversky and Anthony L. Fink
Parkinson’s disease involves the loss of dopaminergic neurons in the substantia nigra, leading to movement disorders. The pathological hallmark of Parkinson’s disease is the presence of Lewy bodies and Lewy neurites, which are intracellular inclusions consisting primarily of alpha-synuclein. Although essentially all cases of sporadic and early-onset Parkinson’s disease are of unknown etiology, two point mutations (A53T and A30P) in the alpha-synuclein gene have been identified in familial early-onset Parkinson’s disease. Previous reports have shown that mutant alpha-synuclein may form fibrils more rapidly than wild-type protein. In order to determine the underlying molecular basis for the enhanced fibrillation of the mutants, the structural properties, responses to changes in the environment, and propensity to aggregate, of wild-type, A30P and A53T alpha-synucleins were systematically investigated. A variety of biophysical methods, including far-UV circular dichroism, FTIR, small-angle X-ray scattering, and light scattering were employed. Neither the natively unfolded, nor the partially folded intermediate conformations, are affected by the familial Parkinson’s disease point mutations. However, both mutants underwent self-association more readily than the wild-type, (i. e. at much lower protein concentration). We attribute this effect to the increased propensity of their partially-folded intermediates to aggregate, rather than to any changes in the monomeric natively unfolded species. This increased propensity of these mutants to aggregate, relative to wild-type alpha-synuclein, would account for the correlation of these mutations with Parkinson’s disease.
Metal-Triggered Structural Transformations, Aggregation and
Fibrillation of Human a-Synuclein.
A Possible Molecular Link Between Parkinson's Disease and Heavy Metal Exposure
J Biol Chem (2001) 276:47, 44284-44297
Vladimir
N. Uversky, Jie Li and Anthony L. Fink
Parkinson's disease (PD) involves the aggregation of a-synuclein to form fibrils, which are the major constituent of intracellular protein inclusions (Lewy bodies and Lewy neurites) in dopaminergic neurons of the substantia nigra. Occupational exposure to specific metals, especially manganese, copper, lead, iron, mercury, zinc, aluminum, appears to be a risk factor for Parkinson's disease based on epidemiological studies. Elevated levels of several of these metals have also been reported in the substantia nigra of PD subjects. We examined the effect of various metals on the kinetics of fibrillation of recombinant a-synuclein and in inducing conformational changes, as monitored by biophysical techniques. Several di- and tri-valent metal ions caused significant accelerations in the rate of a-synuclein fibril formation. Aluminum was the most effective, along with copper (II), iron (III), cobalt (III) and manganese (II). The effectiveness correlated with increasing ion charge density. A correlation was noted between efficiency in stimulating fibrillation and inducing a conformational change, ascribed to formation of a partially-folded intermediate. The potential for ligand bridging by polyvalent metal ions is proposed to be an important factor in the metal-induced conformational changes of a-synuclein. The results indicate that low concentrations of some metals can directly induce a-synuclein fibril formation.
Pesticides directly accelerate the rate of a-synuclein fibril formation: A possible
factor in Parkinson’s disease
FEBS Lett. (2001) 500:3, 105-108
Parkinson’s disease involves intracellular deposits of a-synuclein in the form of Lewy Bodies and Lewy neurites. The etiology of the disease is unknown, however, several epidemiological studies have implicated environmental factors, especially pesticides. Here we show that several pesticides, including rotenone, dieldrin and paraquat, induce a conformational change in a-synuclein and significantly accelerate the rate of formation of a-synuclein fibrils in vitro. We propose that the relatively hydrophobic pesticides preferentially bind to a partially-folded intermediate conformation of a-synuclein, accounting for the observed conformational changes, and leading to association and subsequent fibrillation. These observations suggest one possible underlying molecular basis for Parkinson’s disease.
Probing
the mechanism of insulin fibril formation with insulin mutants
Biochemistry
(2001) 40:28, 8397-8409
Liza Nielsen, Sven Frokjaer, Jens Brange, Vladimir N. Uversky, Anthony L. Fink
The molecular basis of insulin fibril formation was investigated by studying the structural properties and kinetics of fibril formation of 20 different human insulin mutants at both low pH (conditions favoring monomer/dimer) and at pH 7.4 (conditions favoring tetramer/hexamer). Small angle X-ray scattering (SAXS) showed insulin to be monomeric in 20% acetic acid, 0.1M NaCl, pH 2. The secondary structure of the mutants was assessed using far-UV circular dichroism (CD), and the tertiary structure was determined using near-UV CD, quenching of intrinsic fluorescence by acrylamide and interactions with the hydrophobic probe 1-anilino-8-naphthalene-sulfonic acid (ANS). The kinetics of fibril formation were monitored with the fluorescent dye, Thioflavin T (ThT). Neither the degree of insulin self-association, nor any other structural parameter, was found to correlate with the tendency of the insulin mutants to form fibrils. The formation of a partially-folded intermediate as the first step in insulin fibril formation may explain the lack of correlation between the native struc-ture and fibrillation tendencies of the mutants. The results suggest that fibril formation of the insulin mutants is controlled by specific molecular interactions that are sensitive to variations in the primary structure, and the mutations affect the concentration and/or association of a partially-folded intermediate. The marked increase in the lag time for fibril formation with polar mutants demonstrates the importance of hydrophobic interactions in the initial stages of fibrillation.
Ritu
Khurana, Vladimir N. Uversky, Liza Nielsen, and Anthony L. Fink
Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha+beta (lysozyme), beta (Concavalin A) and parallel beta-helical proteins (pectate lyase). Partially-folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent crosslinking and small angle X-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.
Structural and functional similarity
between Yersinia pestis capsular protein Caf1 and human interleukin-1 beta.
Biochemistry. 2001 May 22;40(20):6076-84
Abramov
VM, Vasiliev AM, Vasilenko RN, Kulikova NL, Kosarev IV, Khlebnikov VS,
Ishchenko AT, MacIntyre S, Gillespie JR, Khurana R, Korpela T, Fink AL, Uversky
VN.
Institute of Immunological Engineering, 142380 Lyubuchany, Moscow Region, Russia.
A comparative study of the structural and functional properties of recombinant Yersinia pestis Caf1 and human IL-1beta was performed. According to Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) data, IL-1beta and Caf1 are typical beta-structural proteins. Neither protein interacts with the hydrophobic probe ANS (8-anilino-1-naphthalenesulfonate) under physiological conditions. Specific binding of Caf1 [K(d) = (5.4 +/- 0.1) x 10(-10) M] to interleukin-1 receptors (IL-1Rs) on the surface of finite mouse fibroblasts (line NIH 3T3) was observed. Caf1 is able to inhibit high-affinity binding of (125)I-labeled IL-1beta to NIH 3T3 cells, and in the presence of Caf1, the binding of [(125)I]IL-1beta is characterized by a K(d) of (2.0 +/- 0.3) x 10(-9) M. Caf1 binding to IL-1R could reflect adhesive properties of the capsular subunits responsible for the contact of bacteria with the host immunocompetent cells. In its turn, this may represent a signal for the initiation of the expression and secretion of the proteins of Y. pestis Yop virulon. Thus, these results help to explain the importance of Caf1 in the interaction of Y. pestis with the host immune system.
The effect of environmental factors on the
kinetics of insulin fibril formation: Elucidation of the molecular mechanism
Biochemistry (2001) 40:20, 6036-6046
Liza Nielsen, Ritu Khurana, Alisa Coats, Sven Frokjaer, Jens
Brange, Sandip Vyas, Vladimir N. Uversky, and Anthony L. Fink
In the search for the molecular mechanism of insulin fibrillation, the kinetics of insulin fibril formation were studied under different conditions using the fluorescent dye, Thioflavin T (ThT). The effect of insulin concentration, agitation, pH, ionic strength, anions, seeding, and addition of 1-anilinonaphthalene-8-sulfonic acid (ANS), urea, TMAO, sucrose, and ThT on the kinetics of fibrillation was investigated. The kinetics of the fibrillation process could be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). The addition of seeds eliminated the lag phase. An increase in insulin concentration resulted in shorter lag times and faster growth of fibrils. Shorter lag times and faster growth of fibrils were seen at acidic pH compared to neutral pH, whereas an increase in ionic strength resulted in shorter lag times and slower growth of fibrils. There was no clear correlation between the rate of fibril elongation and ionic strength. Agitation during fibril formation attenuated the effects of insulin concentration and ionic strength on both lag times and fibril growth. The addition of ANS increased the lag time and decreased the apparent growth rate for insulin fibril formation. The ANS-induced inhibition appears to reflect the formation of amorphous aggregates. The denaturant, urea, decreased the lag time, whereas the stabilizers, TMAO and sucrose, increased the lag times. The results indicated that both nucleation and fibril growth were controlled by hydrophobic and electrostatic interactions. A kinetic model, involving the association of monomeric partially-folded intermediates, whose concentration is stimulated by the air-water interface, leading to formation of the critical nucleus and thence fibrils, is proposed.
The Effect of Salts on the Stability and
Folding of Staphylococcal Nuclease
Biochemistry
(2001) 40:7, 2113-2128
Chiaki
Nishimura, Vladimir N. Uversky and Anthony L. Fink
The
stability and folding kinetics of wild-type and a mutant staphylococcal
nuclease (SNase) at neutral pH are significantly perturbed by the presence of
moderate to high concentrations of salts. For the wild-type protein the
presence of sulfate or chloride leads to significantly increased stability
toward thermal and urea denaturation. For example, 0.4 M sodium sulfate
increased the free energy of wild-type SNase by more than 2 kcal/mol. For the
NCA SNase mutant the presence of the salts also abolished the cold denaturation
observed at neutral pH with this variant. For NCA SNase the presence of salt
resulted in a significant increase in secondary and tertiary structure, as
revealed by circular dichroism and intrinsic tryptophan fluorescence, and
increased compactness, as measured by small-angle X-ray scattering.
Substantial effects of salts on the kinetics of refolding were also observed.
For NCA SNase the presence of the salts markedly increased the folding rates
(up to five-fold). On the other hand chloride, in particular, substantially
decreased the rate of folding of the wild-type, although the rates of the slow
phases due to proline isomerization were speeded up. Using fluorescence energy
transfer between the lone tryptophan (Trp140) and an engineered fluorescent
acceptor at residue 64 revealed that the addition of high concentration of KCl
led to the formation of a transient folding intermediate not observed at lower
salt concentrations, and in which residues 140 and 64 were much closer than in
the native state. It is likely that the combination of the high net charge, due
to the high isoelectric point, and the relatively low intrinsic hydrophobicity,
leads to staphylococcal nuclease having only marginal stability at neutral pH.
The salt-induced effects on the structure, stability and kinetics of
staphylococcal nuclease are attributed to the binding of counterions,
especially anions, resulting in minimization of intramolecular charge-charge
(electrostatic) repulsion. This indicates that a localized electrostatic
repulsion occurs at neutral pH even in the wild-type molecule, and that binding
of the counterions leads to screening of the net charge and consequently
reduced repulsion. This in turn leads to increased stability, increased
structure and greater compactness, as observed.
Partially-folded intermediates as critical precursors of light
chain amyloid fibrils and amorphous aggregates
Ritu
Khurana, Joel R. Gillespie, Anupam Talapatra, Lauren J. Minert, Cristian
Ionescu-Zanetti, Ian Millett, and Anthony L. Fink
Light chain, or AL, amyloidosis is a pathological
condition arising from systemic extracellular deposition of monoclonal
immunoglobulin light-chain variable domains in the form of insoluble amyloid
fibrils, especially in the kidneys. Substantial evidence suggests that amyloid
fibril formation from native proteins occurs via a conformational change
leading to a partially-folded intermediate conformation, whose subsequent association
is a key step in fibrillation. In the present investigation we have examined
the properties of a recombinant amyloidogenic light chain variable domain, SMA,
in order to determine whether partially-folded intermediates can be detected
and correlated with aggregation.
The rate of fibril formation from SMA was found to be very sensitive to a
number of extrinsic factors, including pH, protein concentration, ionic
strength, agitation, urea and temperature. The results from spectroscopic and
hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR,
NMR and intrinsic tryptophan fluorescence and small-angle X-ray scattering,
reveal the build-up of two partially-folded intermediate conformational states
as the pH is decreased (low pH destabilized the protein and stimulated the
kinetics of aggregation). A relatively native-like intermediate, IN, was
observed between pH 4 and 6, with little loss of secondary structure, but with
significant tertiary structure changes and enhanced ANS binding, indicating
exposed hydrophobic surfaces. At pH below 3 we observed a relatively unfolded,
but compact, intermediate, IU, which was characterized by decreased tertiary
and secondary structure. The IU intermediate readily forms amyloid fibrils,
whereas IN preferentially leads to amorphous aggregates. Except at pH 2, where
negligible amorphous aggregate is formed, the amorphous aggregates formed
significantly more rapidly than the fibrils. This is the first indication that
different partially-folded intermediates may be responsible for different
aggregation pathways (amorphous and fibrillar). The data support the hypothesis
that amyloid fibril formation involves the ordered self-assembly of
partially-folded species that are critical soluble precursors of fibrils.
Evidence for a partially folded
intermediate in alpha-synuclein fibril formation.
J Biol Chem. 2001 Apr 6;276(14):10737-44.
Epub 2001 Jan 10
Uversky
VN, Li J, Fink AL.
Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of alpha-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that alpha-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant alpha-synuclein. Either a decrease in pH or an increase in temperature transformed alpha-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of alpha-synuclein fibrils. We propose a model for the fibrillation of alpha-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.
Why are "natively unfolded"
proteins unstructured under physiological conditions?
Proteins (2000) 41:3, 415-427
Vladimir N. Uversky, Joel R. Gillespie, Anthony L. Fink
“Natively unfolded” proteins occupy a unique niche within the protein kingdom in that they lack ordered structure under conditions of neutral pH in vitro. Analysis of amino acid sequences, based on the normalized net charge and mean hydrophobicity, has been applied to two sets of proteins: small globular folded proteins and “natively unfolded” ones. The results show that “natively unfolded” proteins are specifically localized within a unique region of charge-hydrophobicity phase space, and indicate that a combination of low overall hydrophobicity and large net charge represent a unique structural feature of “natively unfolded” proteins.
An improved method of preparing the amyloid beta-protein for
fibrillogenesis and neurotoxicity experiments.
Fezoui Y, Hartley DM, Harper JD, Khurana R, Walsh DM, Condron MM, Selkoe DJ, Lansbury PT Jr, Fink AL, Teplow DB.
Center for Neurologic Diseases, Brigham and Women's Hospital, and Department of Neurology, Harvard Medical School, Boston, MA 02215, USA.
Synthetic
amyloid beta-protein (A beta) is used widely to study fibril formation and the
physiologic effects of low molecular weight and fibrillar forms of the peptide
on cells in culture or in experimental animals. Not infrequently, conflicting
results have arisen in these studies, in part due to variation in the starting
conformation and assembly state of A beta. To avoid these problems, we sought a
simple, reliable means of preparing A beta for experimental use. We found that
solvation of synthetic peptide with sodium hydroxide (A beta x NaOH), followed
by lyophilization, produced stocks with superior solubility and fibrillogenesis
characteristics. Solubilization of the pretreated material with neutral buffers
resulted in a pH transition from approximately 10.5 to neutral, avoiding the
isoelectric point of A beta (pI approximately 5.5), at which A beta
precipitation and aggregation propensity are maximal. Relative to trifluoroacetate
(A beta x TFA) or hydrochloric acid (A beta x HCl) salts of A beta, yields of
"low molecular weight A beta" (monomers and/or dimers) were improved
significantly by NaOH pretreatment. Time-dependent changes in circular
dichroism spectra and Congo red dye-binding showed that A beta x NaOH formed
fibrils more readily than did the other A beta preparations and that these
fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for
the preparation of A beta for biophysical and physiologic studies.
Fluorescence energy transfer indicates similar transient and
equilibrium intermediates in staphylococcal nuclease folding.
J Mol Biol. 2000 Jun 16;299(4):1133-46
Nishimura C, Riley R,
Eastman P, Fink AL.
Fluorescence
resonance energy transfer (FRET) is one of the few methods available to measure
the rate at which a folding protein collapses. Using staphylococcal nuclease in
which a cysteine residue was engineered in place of Lys64, permitted FRET
measurements of the distance between the donor tryptophan 140 and
5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled
Cys64. These measurements were undertaken on both equilibrium partially folded
intermediates at low pH (A states), as well as transient intermediates during stopped-flow
refolding. The results indicate that there is an initial collapse of the
protein in the deadtime of the stopped-flow instrument, corresponding to a
regain of approximately 60% of the native signal, followed by three slower
transients. This is in contrast to circular dichroism measurements which show
only 20-25% regain of the native secondary structure in the burst phase. Thus
hydrophobic collapse precedes the formation of substantial secondary structure.
The first two detected transient intermediate species have FRET properties
essentially identical with those of the previously characterized equilibrium A
state intermediates, suggesting similar structures between the equilibrium and
transient intermediates.The effects of anions on the folding of acid-unfolded
staphylococcal nuclease, and urea on the unfolding of the resulting A states,
indicates that in folding the protein becomes compact prior to formation of
major secondary structure, whereas in unfolding the protein expands prior to
major loss of secondary structure. Comparison of the kinetics of refolding of
staphylococcal nuclease, monitored by FRET, and for a proline-free variant,
indicate that folding occurs via two partially folded intermediates leading to
a native-like species with one (or more) proline residues in a non-native
conformation. For the A states an excellent correlation between compactness
measured by FRET, and compactness determined from small-angle X-ray scattering,
was observed. Further, a linear relationship between compactness and free
energy of unfolding was noted. Formation of soluble aggregates of the A states
led to dramatic enhancement of the FRET, consistent with intermolecular
fluorescence energy transfer. Copyright 2000 Academic Press.
Export and folding of signal-sequenceless Bacillus
licheniformis beta-lactamase in Escherichia coli.
Eur J Biochem. 2000 Jun;267(12):3836-47
Frate MC, Lietz EJ, Santos
J, Rossi JP, Fink AL, Ermacora MR.
Departamento de Ciencia y Tecnologia, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina.
Two
genetically engineered variants of the Bacillus licheniformis beta-lactamase
gene were expressed in Escherichia coli. One variant coded for the exo-small
mature enzyme without the signal peptide. The other coded for the exo-large mature
enzyme preceded by 10, mostly polar, residues from an incomplete heterologous
signal. As observed following the extraction by a lysozyme-EDTA treatment, the
signal-less variant was exported to the periplasm with nearly 20% efficiency,
whereas the variant with the N-terminal extension was translocated to a lesser
degree; interestingly, nearly all of the former and half of the latter were
extracted by osmotic shock, which may be of importance for our understanding of
cellular compartments. The fact that a signal-less protein is translocated with
substantial yields raises questions about the essential role of signal peptides
for protein export. As folding and export are related processes, we
investigated the folding in vitro of the two variants. No differences were
found between them. In the absence of denaturant, they are completely folded,
fully active and have a large DeltaG of unfolding. Under partially denaturing
conditions they populate several partially folded states. The absence of
significant amounts of a non-native state under native conditions makes a
thermodynamic partitioning between folding and export less likely. In addition,
kinetic measurements indicated that these B. licheniformis lactamases fold much
faster than E. coli beta-lactamase. This behavior suggests that they are
exported by a kinetically controlled process, mediated by one or more still
unidentified interactions that slow folding and allow a folding intermediate to
enter the export pathway.
Enzyme-induced strain/distortion in the ground-state ES complex in
beta-lactamase catalysis revealed by FTIR
Biochemistry. 2000 May 30;39(21):6538-45
Mark J. Hokenson, Gregory A. Cope, Evan R. Lewis, Keith A. Oberg and Anthony L. Fink
Class A b-lactamases hydrolyze penicillins and other b-lactams via an acyl-enzyme catalytic mechanism. Ser70 is the active site nucleophile. By constructing the S70A mutant, which is unable to form the acyl-enzyme intermediate, it was possible to make stable ES complexes with various substrates. The stability of such Michaelis complexes permitted acquisition of their infrared spectra. Comparison of theb-lactam carbonyl stretch frequency (nCO) in the free and enzyme-bound substrate revealed an average decrease of 13 cm-1, indicating substantial polarization of the lactam carbonyl when bound in the ES complex. Interestingly, regardless of the frequency of the C=O stretch in the free substrate, when complexed to Bacillus licheniformis b-lactamase, the frequency was always 1755±2 cm-1. This suggests the active-site environment induces a similar conformation/polarization of the b-lactam in all substrates when bound to the enzyme. Using deuterium substitution it was shown that factors other than the “oxyanion hole”, which involves H-bonding to two backbone amides, are implicated in the enzyme-induced polarization. The very weak catalytic activity of the S70A b-lactamase suggests enzyme-facilitated hydrolysis due to substrate distortion on binding to the enzyme. Thus the binding of the substrate in the active site induces substantial strain and distortion that contribute significantly to the overall rate enhancement in b-lactamase catalysis.
Lysine-73 is involved in the acylation and deacylation of b-lactamase
Eric. J. Lietz, Heather Truher, Debra Kahn, Mark J. Hokenson and
Anthony L. Fink
Lysine 73 is a conserved active-site residue in the class A b-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the b-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (kcat) and catalytic efficiency (kcat/Km), and a very significant shift in pK1 to higher pH in the bell-shaped pH-rate profiles (kcat/Km) for several penicillin and cephalosporin substrates. The increase in pK1 is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the kcat/Km vs. profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The kcat/Km at the pH optimum for the mutant was down about 200-fold for penicillins and around 104 for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A b-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deacylation. FTIR analysis was used to determine whether acylation or deacylation was rate-limiting. In general, acylation was the rate-limiting step for cephalosporin substrates, whereas deacylation was rate-limiting for penicillin substrates.
Do parallel beta-helix proteins have a
unique fourier transform infrared spectrum?
Khurana R,
Fink AL.
Several
polypeptides have been found to adopt an unusual domain structure known as the
parallel beta-helix. These domains are characterized by parallel beta-strands,
three of which form a single parallel beta-helix coil, and lead to long,
extended beta-sheets. We have used ATR-FTIR (attenuated total
reflectance-fourier transform infrared spectroscopy) to analyze the secondary
structure of representative examples of this class of protein. Because the
three-dimensional structures of parallel beta-helix proteins are unique, we initiated
this study to determine if there was a corresponding unique FTIR signal
associated with the parallel beta-helix conformation. Analysis of the amide I
region, emanating from the carbonyl stretch vibration, reveals a strong
absorbance band at 1638 cm(-1) in each of the parallel beta-helix proteins.
This band is assigned to the parallel beta-sheet structure. However, components
at this frequency are also commonly observed for beta-sheets in many classes of
globular proteins. Thus we conclude that there is no unique infrared signature
for parallel beta-helix structure. Additional contributions in the 1638 cm(-1)
region, and at lower frequencies, were ascribed to hydrogen bonding between the
coils in the loop/turn regions and amide side-chain interactions, respectively.
A 13-residue peptide that forms fibrils and has been proposed to form
beta-helical structure was also examined, and its FTIR spectrum was compared to
that of the parallel beta-helix proteins.
Zn(2+)-mediated structure formation and
compaction of the "natively unfolded" human prothymosin alpha.
Biochem Biophys Res Commun. 2000 Jan
19;267(2):663-8
Uversky
VN, Gillespie JR, Millett IS, Khodyakova AV, Vasilenko RN, Vasiliev AM,
Rodionov IL, Kozlovskaya GD, Dolgikh DA, Fink AL, Doniach S, Permyakov EA,
Abramov VM.
Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia. uversky@hydrogen.ucsc.edu
Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein. Copyright 2000 Academic Press.
Monitoring the Assembly of Immunoglobulin
Light Chain Amyloid Fibrils by Atomic Force Microscopy.
Cristian
Ionescu-Zanetti, Ritu Khurana, Joel R. Gillespie, Jay S. Petrick, Lynne C.
Trabachino, Lauren J. Minert, Sue A. Carter, and Anthony L. Fink
Aggregation of immunoglobulin light chains to form amyloid fibrils is a characteristic feature of AL amyloidosis, a light chain deposition disease. A recombinant variable domain of the light chain, SMA, was used to form amyloid fibrils in vitro. Fibril formation was monitored by atomic force microscopy (AFM) imaging. Single filaments of 2.4 nm diameter were predominant at early times, protofibrils of 4.0 nm in diameter at intermediate times, and Type I and Type II fibrils of 8.0 nm and 6.0 nm in diameter respectively were predominant at the endpoints. The increase in number of fibrils correlated with increased binding of the fluorescent dye Thioflavin T. The fibrils and protofibrils showed a braided structure, suggesting that their formation involves the winding of protofibrils and filaments respectively. This was confirmed by observations that support a model in which two filaments combine to form a protofibril, and two protofibrils intertwine to form a Type I fibril, while three filaments form a Type II fibril.
Transient Dimer
in the Refolding Kinetics of Cytochrome c Characterized by Small Angle
X-Ray Scattering
Biochemistry. 1999
Nov 16;38(46):15352-9
Daniel J. Segel, Vladimir Uversky, Anthony L. Fink, Keith O.
Hodgson and Sebastian Doniach
The equilibrium unfolding and the kinetic
refolding of cytochrome c in the presence of imidazole were studied with small
angle x-ray scattering (SAXS).The equilibrium unfolding experiments showed the
radius of gyration, Rg, of native Cyt c to swell ~1 Å with the
addition of imidazole. The thermodynamic parameter m also reflects an expansion
of the protein as its lower value demonstrates an increase in
solvent-accessible surface area over that of native Cyt c in the absence of
imidazole. Refolding was studied in the presence of imidazole as it prevents
misligated intermediate states from forming during the refolding process,
simplifying the kinetics and making them easier to resolve. Time-resolved
decreases in the forward scattering amplitude, I(0), demonstrated the transient
formation of an aggregated intermediate. Final protein and denaturant
concentrations were varied in the refolding kinetics and the singular value
decomposition (SVD) method was employed to characterize the associated state.
This state was determined to be a dimer, with properties consistent with a
molten globule.
Chaperone-mediated
protein folding.
Physiol Rev. 1999
Apr;79(2):425-49
Anthony L. Fink
The folding of most newly synthesized proteins in the cell requires the interaction of a variety of protein cofactors known as molecular chaperones. These molecules recognize and bind to nascent polypeptide chains and partially-folded intermediates of proteins, preventing their aggregation and misfolding. There are several families of chaperones, those most involved in protein folding are the Hsp40 (DnaJ), Hsp60 (GroEL), and Hsp70 (DnaK) families. The availability of high-resolution structures has facilitated a more detailed understanding of the complex chaperone machinery and mechanisms, including the ATP-dependent reaction cycles of the GroEL and Hsp70 chaperones. For both of these chaperones the binding of ATP triggers a critical conformational change leading to release of the bound substrate protein. Whereas the main role of the Hsp70/Hsp40 chaperone system is to minimize aggregation of newly synthesized proteins, the Hsp60 chaperones also facilitate the actual folding process by providing a secluded environment for individual folding molecules, and may also promote the unfolding and refolding of misfolded intermediates.
Anion-induced
Folding of Staphylococcal Nuclease: Characterization of Multiple
Equilibrium Partially-folded Intermediates.
J
Mol Biol. 1998 May 15;278(4):879-94.
Vladimir N. Uversky, Anton S. Karnoup,
Daniel J. Segel, Sangita Seshadri, Sebastian Doniach, and Anthony L. Fink
The refolding of acid-unfolded staphylococcal nuclease (SNase) induced by anions was characterized, and revealed the existence of three different partially-folded intermediates (A states). The three intermediates lack the rigid tertiary structure characteristic of native states, and differ in their degree of folding as measured by probes of secondary structure, size, stability and globularity. The least structured conformation, A1, is stabilized by chloride (600 mM) or sulfate (100 mM). It is about 50% folded (based on circular dichroism and small angle X-ray scattering (SAXS) data). The next most structured intermediate, A2, is induced by trifluoroacetate (300 mM) and has ~70% native-like secondary structure. The most structured intermediate, A3, is stabilized by trichloroacetate (50 mM) and has native-like secondary structure content and is almost as compact as the native state. The stability toward urea denaturation increases with increasing structure of the intermediates. Moreover, urea-induced unfolding studies show that these partially-folded species are separated from each other, and from the unfolded state, by significant free energy barriers, suggesting that they are distinct conformational states. Kratky plots, based on the SAXS data, indicate that the two more structured intermediates have significant globularity (i. e. a tightly packed core), whereas the less structured intermediate has very little globularity. These observations support a model of protein folding in which certain conformations are of particularly low free energy and hence populated under conditions which differentially destabilize the native state. These partially-folded intermediates probably consist of ensembles of substates with a common core of native-like secondary structure, which is responsible for their stability. Consequently, it is likely that the intermediates observed here represent the equilibrium counterparts of transient kinetic intermediates.
Association-induced
Folding of Globular proteins.
Proc Natl Acad
Sci U S A. 1998 May 12;95(10):5480-3
Vladimir N. Uversky, Daniel J. Segel, Sebastian Doniach and Anthony L. Fink
It has generally been assumed that the aggregation of partially-folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially-folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in a monomeric, partially-folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations. In addition, more globularity, as determined from Kratky plots of small-angle X-ray scattering data, was also noted in the associated states.
ASSOCIATION OF PARTIALLY-FOLDED INTERMEDIATES
OF STAPHYLOCOCCAL NUCLEASE INDUCES STRUCTURE AND STABILITY.
Protein Science (1999) 8, 161-173
Vladimir N.
Uversky, Anton S. Karnoup, Ritu Khurana, Daniel J. Segel, Sebastian Doniach and
Anthony L. Fink
Staphylococcal nuclease forms three different partially-folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability and compactness. Although these intermediates are monomeric at low protein concentration (< 0.25 mg/ml), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability and globularity of the two less-ordered partially-folded intermediates (A1 and A2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, FTIR and small-angle X-ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state.
"Natively Unfolded" Human
Prothymosin alpha Adopts Partially-Folded Collapsed Conformation at
Acidic pH.
Biochemistry (1999) 38, 15009-15016
Vladimir N.
Uversky, Joel R. Gillespie, Ian S. Millet, Anna V. Khodyakova, Anatoly M.
Vasiliev, Tatyana V. Chernovskaya, Raisa N. Vasilenko, Galina D. Kozlovskaya,
Dmitry A. Dolgikh, Anthony L. Fink, Sebastian Doniach andVyacheslav M. Abramov
Prothymosin has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin were characterized at neutral and acidic pH by gel-filtration, SAXS, circular dichroism, ANS fluorescence, 1H-NMR and resistance to urea-induced unfolding. Interestingly, prothymosin underwent a cooperative transition from the unfolded state into a partially-folded conformation on lowering the pH. This conformation of prothymosin is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid–rich elements of the protein, accompanied by the partial hydrophobic collapse, is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.
Do Parallel b-Helix Proteins have a Unique FTIR
Spectrum?
Biophysical
Journal (2000) 78, 994-1000
Ritu Khurana and Anthony L. Fink
Several polypeptides have been found to adopt an unusual domain structure known as the parallel b-helix. These domains are characterized by parallel b-strands, three of which form a single parallel b-helix coil, and lead to long extended b-sheets. We have used ATR-FTIR (Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy) to analyze the secondary structure of representative examples of this class of protein. Since the three-dimensional structures of parallel b-helix proteins are unique, we initiated this study to determine if there was a unique FTIR signal associated with the parallel b-helix conformation. Analysis of the amide I region, emanating from the carbonyl stretch vibration, reveals a strong absorbance band at 1638 cm-1 in each of the parallel b-helix proteins. This band is assigned to the parallel b-sheet structure. However, components at this frequency are also commonly observed for b-sheets in many classes of globular proteins. Thus, we conclude that there is no unique infrared signature for parallel b-helix structure. Additional contributions in the 1638 cm-1 region, and at lower frequencies, were ascribed to hydrogen-bonding between the coils in the loop/turn regions and amide side-chain interactions, respectively. A 13-residue peptide that forms amyloid fibrils and has been proposed to form b-helical structure was also examined and its FTIR spectrum compared to that of the parallel b-helix proteins.
PROTEIN AGGREGATION: Folding aggregates,
inclusion bodies and amyloid.
Folding & Design (1998) 3, R9-R23
Anthony L. Fink
The importance of protein aggregation in a wide variety of situations is now gaining its due recognition. The focus of this review is on aggregation resulting in the formation of inclusion bodies, amyloid fibrils and folding aggregates. Substantial data support the hypothesis that partially-folded intermediates are key precursors to aggregates, that aggregation involves specific intermolecular interactions and that most aggregates involve b-sheet.
Discrete intermediates vs. molten globule
models of protein folding: Characterization of partially-folded intermediates
of apomyoglobin.
Folding & Design (1997) 3, 19-25
Background: Although small proteins may fold in an apparent two-state manner, most studies of protein folding reveal transient intermediates, and the “molten globule” has been proposed to be a general intermediate in protein folding. Relatively little is known about the structure of partially-folded intermediates.
Results: Three different partially-folded intermediates of apomyoglobin were characterized at low pH in the presence of different anions, having 35%, 50% and 60% helix. Increasing helical structure correlated with decreasing size and increasing stability to urea. Similar intermediates have been observed transiently during the folding of apomyoglobin.
Conclusions: The results are consistent with a model for folding in which structural units coalesce to form a core of relatively native-like structure, the remainder of the protein being relatively disordered. For a given protein there will be certain partially-folded conformations of particularly low free energy, which are preferentially populated under both equilibrium and transient folding conditions. The conformation/topology of the intermediates will be specific to a given protein, i. e. there are no "general" intermediates, such as the molten globule, in folding.
Determination of
Secondary Structure in Protein Aggregates Using Attenuated Total Reflectance
(ATR) FTIR.
Infrared Analysis
of Peptides and Proteins: Principles and Applications, Bal Ram Singh,
Editor. Washington, DC, American Chemical Society, c2000. Series title: ACS
Symposium Series 750.
A. L. Fink, S. Seshadri, R. Khurana, and K. A. Oberg
Protein aggregation is a major problem in many fields. Currently the mechanism of formation and the structure of protein aggregates are poorly understood. The insoluble nature of protein aggregates limits the number of techniques which can be used to ascertain the conformation of the aggregated protein. Attenuated total reflectance (ATR) FTIR is one method which can readily provide information about the secondary structure content of both soluble and insoluble proteins. ATR-FTIR was used to examine the structure of inclusion bodies, folding aggregates, amorphous precipitates and amyloid fibrils. A common feature of the aggregated proteins is the presence of additional b-structure compared to the native conformation.