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.
Maiti
Nakul C
NC
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Apetri
Mihaela M
MM
Zagorski
Michael G
MG
Carey
Paul R
PR
Anderson
Vernon E
VE
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