Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein.
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.
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Nakul C
NC
Maiti
Mihaela M
MM
Apetri
Michael G
MG
Zagorski
Paul R
PR
Carey
Vernon E
VE
Anderson
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry; D002364:Caseins / Q000737:chemistry; D002942:Circular Dichroism; D006801:Humans; D000432:Methanol / Q000737:chemistry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010774:Phosvitin / Q000737:chemistry; D020005:Propanols / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012996:Solutions; D013059:Spectrum Analysis, Raman / Q000379:methods*; D051843:Synucleins; D051844:alpha-Synuclein; D006845:Hydrocarbons, Fluorinated
D000577:Amides; D000682:Amyloid; D002364:Caseins; D000432:Methanol; D009419:Nerve Tissue Proteins; D010774:Phosvitin; D020005:Propanols; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D012996:Solutions; D051843:Synucleins; D051844:alpha-Synuclein; C497604:SNCA protein, human; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated