Raman spectroscopic characterization of secondary structure in natively unfolded proteins:: α-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 a-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 a-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 similar to33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of similar to26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of a-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 a-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-Abeta 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.