Characterization of oligomeric intermediates in alpha-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-synuclein

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.     

Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers → monomers → partially unfolded monomers → oligomeric aggregates → fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.     

Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the β-sheet (up to 26%) and unordered conformation at the expense of α-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils can be formed by an antiparallel arrangement of β-strands forming the β-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures, laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align within the immediate vicinity of the fibers. This filament + monomers/oligomers scenario is an alternative pathway to the otherwise dominant filament + filament manner of the protein fibril's lateral growth. Conformational preferences for a certain pathway to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered.     

UV Resonance Raman explores protein structural modification upon fibrillation and ligand interaction

Amyloids are proteinaceous deposits considered an underlying pathological hallmark of several degenerative diseases. The mechanism of amyloid formation and its inhibition still represent challenging issues, especially when protein structure cannot be investigated by classical biophysical techniques as for the intrinsically disordered proteins (IDPs). In this view, the need to find an alternative way for providing molecular and structural information regarding IDPs prompted us to set a novel, to our knowledge, approach focused on UV Resonance Raman (UVRR) spectroscopy. To test its applicability, we study the fibrillation of hen-egg white lysozyme (HEWL) and insulin as well as their interaction with resveratrol, employing also intrinsic fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). The increasing of the β-sheet structure content at the end of protein fibrillation probed by FTIR occurs simultaneously with a major solvent exposure of tryptophan (Trp) and tyrosine (Tyr) residues of HEWL and insulin, respectively, as revealed by UVRR and intrinsic fluorescence spectroscopy. However, because the latter technique is successfully used when proteins naturally contain Trp residues, it shows poor performances in the case of insulin, and the information regarding its tertiary structure is exclusively provided by UVRR spectroscopy. The presence of an increased concentration of resveratrol induces mild changes in the secondary structure of both protein fibrils while remodeling HEWL fibril length and promoting the formation of amorphous aggregates in the case of insulin. Although the intrinsic fluorescence spectra of proteins are hidden by resveratrol signal, UVRR Trp and Tyr bands are resonantly enhanced, showing a good sensitivity to the presence of resveratrol and marking a modification in the noncovalent interactions in which they are involved. Our findings demonstrate that UVRR is successfully employed in the study of aggregation-prone proteins and of their interaction with ligands, especially in the case of Trp-lacking proteins.     

Multiple tryptophan probes reveal that ubiquitin folds via a late misfolded intermediate

Much of our understanding of protein folding mechanisms is derived from experiments using intrinsic fluorescence of natural or genetically inserted tryptophan (Trp) residues to monitor protein refolding and site-directed mutagenesis to determine the energetic role of amino acids in the native (N), intermediate (I) or transition (T) states. However, this strategy has limited use to study complex folding reactions because a single fluorescence probe may not detect all low-energy folding intermediates. To overcome this limitation, we suggest that protein refolding should be monitored with different solvent-exposed Trp probes. Here, we demonstrate the utility of this approach by investigating the controversial folding mechanism of ubiquitin (Ub) using Trp probes located at residue positions 1, 28, 45, 57, and 66. We first show that these Trp are structurally sensitive and minimally perturbing fluorescent probes for monitoring folding/unfolding of the protein. Using a conventional stopped-flow instrument, we show that ANS and Trp fluorescence detect two distinct transitions during the refolding of all five Trp mutants at low concentrations of denaturant: T₁, a denaturant-dependent transition and T₂, a slower transition, largely denaturant-independent. Surprisingly, some Trp mutants (Ub^M1W, Ub^S57W) display Trp fluorescence changes during T₁ that are distinct from the expected U → N transition suggesting that the denaturant-dependent refolding transition of Ub is not a U → N transition but represents the formation of a structurally distinct I-state (U → I). Alternatively, this U → I transition could be also clearly distinguished by using a combination of two Trp mutations Ub^F45W-T66W for which the two Trp probes that display fluorescence changes of opposite sign during T₁ and T₂ (Ub^F45W-T66W). Global fitting of the folding/unfolding kinetic parameters and additional folding-unfolding double-jump experiments performed on Ub^M1W, a mutant with enhanced fluorescence in the I-state, demonstrate that the I-state is stable, compact, misfolded, and on-pathway. These results illustrate how transient low-energy I-states can be characterized efficiently in complex refolding reactions using multiple Trp probes.     

Steady-state and time-resolved fluorescence studies of the intestinal fatty acid binding protein

The intestinal fatty acid binding protein contains two tryptophan residues (Trp6 and Trp82) both of which have been shown by X-ray and NMR methods to be buried in hydrophobic clusters. By using a combination of steady-state and time-resolved fluorescence experiments, we have deconvoluted the lifetime weighted contribution of each of the tryptophans to the steady-state fluorescence quantum yield. While Trp82 has been implicated in an intermediate that appears at relatively high denaturant concentrations, the variation of the lifetime weighted contribution of Trp6 with urea or guanidium hydrochloride shows formation of an intermediate state at low concentrations of the denaturant before the actual unfolding starts. Trp82 did not show similar behavior. Fluorescence quenching experiments by acrylamide show that while Trp6 in the native protein is less solvent-exposed, its accessibility is increased significantly at low urea concentration indicating that the early intermediate state is partially unfolded. Time-resolved anisotropy experiments indicate that the volume of the partially unfolded intermediates is larger than the native protein and lead to the speculation that the last step of the protein folding might be the removal of solvent molecules from the protein.     

Surface expansion is independent of and occurs faster than core solvation during the unfolding of barstar

The denaturant-induced unfolding kinetics of the 89-residue protein, barstar, have been examined using fluorescence resonance energy transfer (FRET) at 25 degrees C and pH 8.0. The core tryptophan, Trp53, in barstar serves as a fluorescence donor, and a thionitrobenzoic acid moiety (TNB) attached to a cysteine residue acts as an acceptor to form an efficient FRET pair. Four different single-cysteine containing mutants of barstar with cysteine residues at positions 25, 40, 62, and 82 were studied. The unfolding kinetics of the four mutant forms of barstar were monitored by measurement of the changes in the fluorescence intensity of Trp53 in the unlabeled and TNB-labeled proteins. The rate of change of fluorescence of the single-tryptophan residue, Trp53, in the unlabeled protein, where no FRET occurs, yields the rate of solvation of the core. This rate is similar for all four unlabeled proteins. The rate of the increase in the fluorescence of Trp53 in the labeled protein, where FRET from the tryptophan to the TNB label occurs, yields the rate of decrease in FRET efficiency during unfolding. The decrease in FRET efficiency for proteins labeled at either of the two buried positions (Cys40 or Cys82) occurs at a rate similar to the rate of core solvation. The decrease in FRET efficiency for the acceptor at Cys40 is also shown to be sensitive to the isomerization of the Tyr47-Pro48 cis bond. For the proteins where the label is at a solvent-exposed position (Cys25 and Cys62), the decrease in FRET efficiency occurs in two kinetic phases; 15-25% of the FRET efficiency decreases in the faster phase, and the remaining FRET efficiency decreases in a slower phase, the rate of which is the same as the rate of core solvation. These results clearly indicate that, during unfolding, the protein surface expands faster than, and independently of, water intrusion into the core.     

Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

Impact of Tyr to Ala mutations on alpha-synuclein fibrillation and structural properties

Substantial evidence suggests that the fibrillation of alpha-synuclein is a critical step in the development of Parkinson's disease. In vitro, alpha-synuclein forms fibrils with morphologies and a staining characteristic similar to those extracted from disease-affected brain. Monomeric alpha-synuclein is an intrinsically disordered protein, with three Tyr residues in the C-terminal region, one in the N-terminus, and lacking Trp. It is thought that interactions between the C-terminus and the central portion of the molecule may prevent or minimize aggregation/fibrillation. To test this hypothesis we examined the importance of the Tyr residues on the propensity for alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited, in the timescale over which measurements were made, by replacing the three C-terminal Tyr residues with Ala. In addition, substitution of Tyr133 by Ala also resulted in the absence of fibrillation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibrillation. Structural analysis showed that the Y133A mutant had a substantially different conformation, rich in alpha-helical secondary structure, as compared with the wild-type and other mutants, although the formation of any tertiary structure has not been observed as can be judged from near-UV-CD spectra. These observations suggest that the long-range intramolecular interactions between the N- and C-termini of alpha-synuclein are likely to be crucial to the fibrillation process.     

Tryptophan in alpha-helix 3 of Tet repressor forms a sequence-specific contact with tet operator in solution

The Tn10-encoded Tet repressor contains two tryptophan residues at positions 43 and 75. The typical tryptophan fluorescence is decreased upon binding of tet operator. The Tet repressor gene was engineered to replace either or both of the Trp codons by Phe codons. The resulting single tryptophan mutants are called F43 and F75 and the double mutant F43F75. The mutant proteins were purified to homogeneity. They recognize tet operator DNA only in the absence of the inducer tetracycline, indicating an intact tertiary structure of the engineered proteins. F75 and wild-type bind tet operator with the same association constant. The association constants of F43 and F43F75 with tet operator are about 3 orders of magnitude smaller. This indicates that Trp43 is important for tet operator recognition. Trp43 fluorescence is completely quenched in the complex with tet operator DNA while Trp75 remains unaffected. Binding to nonspecific DNA leads only to a 40% decrease of Trp43 fluorescence. This is interpreted as the contribution of the changed environment while the complete quench reflects a tight sequence-specific contact of tryptophan 43 to tet operator DNA. Trp43 is solvent-exposed, while Trp75 is buried in the hydrophobic interior of the protein. These results are discussed in light of the alpha-helix turn-alpha-helix DNA binding motif deduced from homology to other repressor proteins.     

Spectroscopic Characterization of Arrestin Interactions with Competitive Ligands: Study of Heparin and Phytic Acid Binding

A combination of intrinsic fluorescence and circular dichroic (CD) spectroscopy has been used to characterize the complexes formed between bovine retinal arrestin and heparin or phytic acid, two ligands that are known to mimic the structural changes in arrestin attending receptor binding. No changes in the CD spectra were observed upon ligand binding, nor did the degree of tryptophan fluorescence quenching change significantly in the complexes. These data argue against any large-scale changes in protein secondary or tertiary structure accompanying ligand binding. The change in tyrosine fluorescence intensity was used to determine the dissociation constants for the heparin and phytic acid complexes of arrestin. The only change observed was a saturable diminution of tyrosine fluorescence signal from the protein. For both ligands, the data suggest two distinct binding interactions with the protein—a high-affinity interaction with K _d between 200 and 300 nM, and a lower affinity interaction with K _d between 2 and 8 M. Study of collisional quenching of tyrosine fluorescence in free arrestin and the ligand-replete complexes indicates that 10 of the 14 tyrosine residues of the protein are solvent-exposed in the free protein; this value drops to between 5 and 6 solvent-exposed residues in the high-affinity complexes of the two ligands. These data suggest that ligand binding leads to direct occlusion of between 4 and 5 tyrosine residues on the solvent-exposed surface of the protein, but not to any large-scale changes in protein structure. The large activation energy previously reported to be associated with arrestin–receptor interactions may therefore reflect localized movements of the N- and C-termini of arrestin, which are proposed to interact in the free protein through electrostatic interactions. Binding of the anionic ligands heparin, phytic acid, or phosphorylated rhodopsin may compete with the C-terminus of arrestin for these electrostatic interactions, thus allowing the C-terminus to swing out of the binding region.     

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.     

Probing a pheromone binding protein of the silkmoth Antheraea polyphemus by endogenous tryptophan fluorescence.

One subtype of the pheromone binding proteins of the silkmoth Antheraea polyphemus (ApolPBP1) has been analysed exploiting the two endogenous tryptophan residues as fluorescent probe. The intrinsic fluorescence exhibited a rather narrow spectrum with a maximum at 336 nm. Site-directed mutagenesis experiments revealed that one of the tryptophan residues (Trp37) is located in a hydrophobic environment whereas Trp127 is more solvent exposed, as was predicted modeling the ApolPBP1 sequence on the proposed structure of the Bombyx mori pheromone binding protein. Monitoring the interaction of ApolPBP1 as well as its Trp mutants with the three species-specific pheromone compounds by recording the endogenous fluorescence emission revealed profound differences; whereas (E6,Z11)-hexadecadienal induced a dose-dependent quenching of the fluorescence, both (E6,Z11)-hexadecadienyl-1-acetate and (E4,Z9)-tetradecadienyl-1-acetate elicited an augmentation of the endogenous fluorescence. These data indicate that although ApolPBP1 can bind all three pheromones, there are substantial differences concerning their interaction with the protein, which may have important functional implications.     

DNA induces folding in alpha-synuclein: understanding the mechanism using chaperone property of osmolytes

Alpha-synuclein conformational modulation leading to fibrillation has been centrally implicated in Parkinson's disease. Previously, we have shown that alpha-synuclein has DNA binding property. In the present study, we have characterized the effect of DNA binding on the conformation and fibrillation kinetics of alpha-synuclein. It was observed that single-stranded circular DNA induce alpha-helix conformation in alpha-synuclein while plasmid supercoiled DNA has dual effect inducing a partially folded conformation and alpha-helix under different experimental conditions. Interestingly, alpha-synuclein showed a specificity for GC* nucleotide sequence in its binding ability to DNA. The aggregation kinetics data showed that DNA which induced partially folded conformation in alpha-synuclein promoted the fibrillation while DNA which induced alpha-helix delayed the fibrillation, indicating that the partially folded intermediate conformation is critical in the aggregation process. Further, the mechanism of DNA-induced folding/aggregation of alpha-synuclein was studied using effect of osmolytes on alpha-synuclein as a model system. Among the five osmolytes used, Glycerol, trimethylamine-N-oxide, Betaine, and Taurine induced partially folded conformation and in turn enhanced the aggregation of alpha-synuclein. The ability of DNA and osmolytes in inducing conformational transition in alpha-synuclein, indicates that two factors are critical in modulating alpha-synuclein folding: (i) electrostatic interaction as in the case of DNA, and (ii) hydrophobic interactions as in the case of osmolytes. The property of DNA inducing alpha-helical conformation in alpha-synuclein and inhibiting the fibrillation may be of significance in engineering DNA-chip based therapeutic approaches to PD and other amyloid disorders.     

Evidence for the involvement of tryptophan 38 in the active site of glutathione S-transferase P.

Glutathione S-transferase P (GST-P) exists as a homodimeric form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modification by N-bromosuccinimide (NBS). The quenching of intrinsic fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost when single tryptophan residue was oxidized by NBS. To identify which tryptophan residue is involved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activity with that of the wild type one. Trp38His mutant neither was bound to S-hexylglutathione-linked Sepharose nor exhibited any GST activity. These findings indicate that Trp38 is important for the catalytic function and substrate binding of GST-P.     

Rational discovery of a SOD1 tryptophan oxidation inhibitor with therapeutic potential for amyotrophic lateral sclerosis

Abstract

Formation of Cu, Zn superoxide dismutase 1 (SOD1) protein inclusions within motor neurons is one of the principal characteristics of SOD1-related amyotrophic lateral sclerosis (ALS). A hypothesis as to the nature of SOD1 aggregation implicates oxidative damage to a solvent-exposed tryptophan as causative. Here, we chart the discovery of a phenanthridinone based compound (Lig9) from the NCI Diversity Set III by rational methods by in silico screening and crystallographic validation. The crystal structure of the complex with SOD1, refined to 2.5 Å, revealed that Lig9 binds the SOD1 β-barrel in the β-strand 2 and 3 region which is known to scaffold SOD1 fibrillation. The phenanthridinone moiety makes a substantial π–π interaction with Trp32 of SOD1. The compound possesses a significant binding affinity for SOD1 and inhibits oxidation of Trp32; a critical residue for SOD1 aggregation. Thus, Lig9 is a good candidate from which to develop a new library of SOD1 aggregation inhibitors through protection of Trp32 oxidation.

Communicated by Ramaswamy H. Sarma     

Anti-oligomeric single chain variable domain antibody differentially affects huntingtin and alpha-synuclein aggregates

Huntington's and Parkinson's diseases are both neurodegenerative disorders caused at least in part by misfolding and aggregation of huntingtin (htt) and alpha-synuclein, respectively. Here we use a single chain antibody fragment (scFv) isolated against oligomeric alpha-synuclein to probe similarities and differences between the aggregation and toxic mechanisms of htt and alpha-synuclein. When incubated with htt, the scFv both blocks formation of and promotes dissociation of fibrillar aggregates, but stabilizes formation of cytotoxic oligomeric aggregates. Previous studies with monomeric alpha-synuclein showed the scFv prevented fibrillar aggregation, but blocked toxicity of oligomeric aggregates. These divergent effects suggest the toxic mechanisms of oligomeric aggregates differ among amyloidogenic protein species.     

Metal-induced changes in the fluorescence properties of tyrosine and tryptophan site-specific mutants of oncomodulin.

Oncomodulin is a 108-residue, oncodevelopmental protein containing two calcium-binding sites identified as the CD- and EF-loops. The protein contains no tryptophan and only two tyrosine residues, one which is a calcium ligand in the CD-loop (Tyr-57) and one which lies in the flanking D-helix of this loop (Tyr-65). Site-specific mutagenesis was performed to yield five mutants, two with phenylalanine substituted for tyrosine in positions 57 and 65 and three with tryptophan substituted into position 57 in the CD-loop, position 65 in the D-helix, and position 96 in the EF-loop. The single Tyr-containing mutants demonstrated that position 57 was perturbed to a significantly greater extent than position 65 upon calcium binding. Although both tyrosine residues responded to decalcification, the fluorescence intensity changes were in opposite directions, with the more dominant Tyr-57 accounting for the majority of the intrinsic fluorescence observed in native oncomodulin. The substitution of tryptophan for each tyrosyl residue revealed that in both positions the tryptophan resided in polar, conformationally heterogeneous environments. The environment of Trp-57 was affected by Ca2+ binding to a much greater extent compared to that of Trp-65. Only 1 equiv of Ca2+ was required to produce greater than 70% of the Trp fluorescence changes in positions 57 and 65, indicating that Ca2+ binding to the higher affinity EF-loop had a pronounced effect on the protein structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observation of multiple intermediates in alpha-synuclein fibril formation by singular value decomposition analysis

One of the most well known characteristics for Parkinson's disease (PD) is a polymerization of wild-type or mutant alpha-synuclein into aggregates and fibrils, commonly observed as Lewy bodies and Lewy neuritis in PD patients. Although numerous studies on alpha-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation are not well understood yet. In the present study, structural properties and propensities to form fibrils of wild-type, A30P, E46K, and A53T alpha-synucleins were investigated using fluorescence and circular dichroism (CD) methods. The results from these studies were analyzed using singular value decomposition (SVD) method which estimates a number of conformationally independent species for a given process. The time-dependent CD spectra of the wild-type alpha-synuclein indicated a multi-step process in the fibril formation, and SVD analysis using the time-dependent CD spectra revealed that five or nine intermediates were formed at the early stage of fibrillation.     

Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state

Aggregation of partially folded intermediates populated during protein folding processes has been described for many proteins. Likewise, partially unfolded chains, generated by perturbation of numerous proteins by heat or chemical denaturants, have also been shown to aggregate readily. However, the process of protein aggregation from native-state conditions is less well understood. Granulocyte-colony stimulating factor (G-CSF), a member of the four-helix bundle class of cytokines, is a therapeutically relevant protein involved in stimulating the growth and maturation of phagocytotic white blood cells. Under native-like conditions (37 degrees C [pH 7.0]), G-CSF shows a significant propensity to aggregate. Our data suggest that under these conditions, native G-CSF exists in equilibrium with an altered conformation, which is highly aggregation prone. This species is enriched in 1-2 M GdmCl, as determined by tryptophan fluorescence and increased aggregation kinetics. In particular, specific changes in Trp58 fluorescence report a local rearrangement in the large loop region between helices A and B. However, circular dichroism, reactivity toward cyanylation, and ANS binding demonstrate that this conformational change is subtle, having no substantial disruption of secondary and tertiary structure, reactivity of the free sulfhydryl at Cys17 or exposure of buried hydrophobic regions. There is no indication that this altered conformation is important to biological activity, making it an attractive target for rational protein stabilization.