alpha-synuclein and hexafluoroisopropanol

The Parkinson's protein alpha-synuclein binds to synaptic vesicles in vivo and adopts a highly extended helical conformation when binding to lipid vesicles in vitro. High-resolution structural analysis of alpha-synuclein bound to small lipid or detergent micelles revealed two helices connected by a non-helical linker, but corresponding studies of the vesicle-bound extended-helix state are hampered by the size and heterogeneity of the protein-vesicle complex. Here we employ fluorinated alcohols (FAs) to induce a highly helical aggregation-resistant state of alpha-synuclein in solution that resembles the vesicle-bound extended-helix state but is amenable to characterization using high-resolution solution-state NMR. Analysis of chemical shift, NOE, coupling constant, PRE and relaxation measurements shows that the lipid-binding domain of alpha-synuclein in FA solutions indeed adopts a single continuous helix and that the ends of this helix do not come into detectable proximity to each other. The helix is well ordered in the center, but features an increase in fast internal motions suggestive of helix fraying approaching the termini. The central region of the helix exhibits slower time scale motions that likely result from flexing of the highly anisotropic structure. Importantly, weak or missing short- and intermediate-range NOEs in the region corresponding to the non-helical linker of micelle-bound alpha-synuclein indicate that the helical structure in this region of the protein is intrinsically unstable. This suggests that conversion of alpha-synuclein from the extended-helix to the broken-helix state represents a functionally relevant structural transition.

Topics: Alcohols; alpha-Synuclein; Humans; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Parkinson Disease; Propanols; Protein Aggregates; Protein Structure, Secondary; Trifluoroethanol

Assembly of amyloidogenic proteins into toxic oligomers and fibrils is an important pathogenic feature of over 30 amyloid-related diseases. Understanding the structures and mechanisms involved in the assembly process is necessary for rational approaches geared at inhibiting formation of these toxic species. Here, we review the application of photo-induced cross-linking of unmodified proteins (PICUP) to two disease-related amyloidogenic proteins (1) islet amyloid polypeptide (IAPP), whose toxic oligomers are thought to cause the demise of pancreatic β-cells in type-2 diabetes mellitus and (2) α-synuclein, which aggregates into toxic oligomers and precipitates in Lewy bodies in Parkinson's disease. PICUP is an effective method allowing chemical "freezing" of dynamically changing oligomers and subsequent study of the oligomer size distribution that existed before cross-linking. The method has provided insights into the factors controlling early oligomerization, which could not be obtained by other means. We discuss sample preparation, experimental details, optimization of parameters, and troubleshooting.

Topics: alpha-Synuclein; Electrophoresis, Polyacrylamide Gel; Islet Amyloid Polypeptide; Photochemical Processes; Propanols; Protein Multimerization; Protein Structure, Secondary; Silver Staining; Solubility

The intrinsically disordered protein α-synuclein aggregates into amyloid fibrils, a process known to be implicated in several neurodegenerative states. Partially folded forms of the protein are thought to trigger the aggregation process. Here, α-synuclein conformers are characterized by analysis of the charge-state distributions observed in electrospray-ionization mass spectrometry under negative-ion mode. It is found that, even at neutral pH, a small fraction of the molecular population is in a compact conformation. Several distinct partially folded forms are then identified under conditions that promote α-synuclein aggregation, such as solutions of simple and fluorinated alcohols. Specific intermediates accumulate at increasing concentrations of ethanol, hexafluoro-2-propanol, and trifluoroethanol. Finally, extensive folding induced by Cu(2+) binding is revealed by titrations in the presence of Cu(2+)-glycine. The data confirm the existence of a single, high-affinity binding site for Cu(2+). Because accumulation of this partially folded form correlates with enhancement of fibrillation kinetics, it is likely to represent an amyloidogenic intermediate in α-synuclein conformational transitions.

Topics: alpha-Synuclein; Copper Sulfate; Hydrogen-Ion Concentration; Methanol; Propanols; Protein Folding; Protein Structure, Tertiary; Spectrometry, Mass, Electrospray Ionization; Trifluoroethanol

Human alpha-synuclein is the causative protein of several neurodegenerative diseases, such as Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). The N-terminal half of alpha-synuclein contains seven imperfect repeat sequences. One of the PD/DLB-causing point mutations, E46K, has been reported in the imperfect repeat sequences of alpha-synuclein, and is prone to form amyloid fibrils. The presence of seven imperfect repeats in alpha-synuclein raises the question of whether or not mutations corresponding to E46K in the other imperfect KTKE(Q)GV repeats have similar effects on aggregation and fibrillation, as well as their propensities to form alpha-helices. To investigate the effect of E(Q)/K mutations in each imperfect repeat sequence, we substituted the amino acid corresponding to E46K in each of the seven repeated sequences with a Lys residue. The mutations in the imperfect KTKE(Q)GV repeat sequences of the N-terminal region were prone to decrease the lag time of fibril formation. In addition, AFM imaging suggested that the Q24K mutant formed twisted fibrils, while the other mutants formed spherical aggregates and short fibrils. These observations indicate that the effect of the mutations on the kinetics of fibril formation and morphology of fibrils varies according to their location.

Topics: alpha-Synuclein; Amino Acid Sequence; Amino Acid Substitution; Circular Dichroism; Humans; Light; Microscopy, Atomic Force; Molecular Sequence Data; Mutant Proteins; Propanols; Protein Structure, Quaternary; Protein Structure, Secondary; Repetitive Sequences, Amino Acid; Scattering, Radiation

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.

Topics: alpha-Synuclein; Amides; Amino Acid Sequence; Amyloid; Caseins; Circular Dichroism; Humans; Methanol; Molecular Sequence Data; Nerve Tissue Proteins; Phosvitin; Propanols; Protein Conformation; Protein Folding; Protein Structure, Secondary; Recombinant Proteins; Sodium Dodecyl Sulfate; Solutions; Spectrum Analysis, Raman; Synucleins

The conversion of alpha-synuclein into amyloid fibrils in the substantia nigra is linked to Parkinson's disease. Alpha-synuclein is natively unfolded in solution, but can be induced to form either alpha-helical or beta-sheet structure depending on its concentration and the solution conditions. The N-terminus of alpha-synuclein comprises seven 11-amino acid repeats (XKTKEGVXXXX) which can form an amphipathic alpha-helix. Why seven repeats, rather than six or eight, survived the evolutionary process is not clear. To probe this question, two sequence variants of alpha-synuclein, one with two fewer (del2) and one with two additional (plus2) repeats, were studied. As compared to wild-type alpha-synuclein, the plus2 variant disfavors the formation of beta-sheet-rich oligomers, including amyloid fibrils. In contrast, the truncated variant, del2, favors beta-sheet and fibril formation. We propose that the repeat number in WT alpha-synuclein represents an evolutionary balance between the functional conformer of alpha-synuclein (alpha-helix and/or random coil) and its pathogenic beta-sheet conformation. N-terminal truncation of alpha-synuclein may promote pathogenesis.

Topics: alpha-Synuclein; Amino Acid Sequence; Amyloid; Circular Dichroism; Cloning, Molecular; Humans; Molecular Sequence Data; Nerve Tissue Proteins; Parkinson Disease; Peptide Fragments; Propanols; Protein Folding; Protein Isoforms; Protein Structure, Secondary; Protein Structure, Tertiary; Repetitive Sequences, Amino Acid; Synucleins

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.

Topics: Alcohols; alpha-Synuclein; Benzothiazoles; Circular Dichroism; Ethanol; Fluorescent Dyes; Humans; Kinetics; Light; Models, Chemical; Nerve Tissue Proteins; Phospholipids; Propanols; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Scattering, Radiation; Solvents; Spectroscopy, Fourier Transform Infrared; Synucleins; Thiazoles; Trifluoroethanol