[14] Protein secondary structure analysis using Raman amide I and amide III spectra

This chapter describes methods for the estimation of protein secondary structure content—in terms of percentage helix, β-strand, and reverse turn—from a least-squares analysis of Raman amide I and amide III spectra. A statistical analysis of these estimates for proteins with known structures is included to establish the degree of confidence that may be placed on results for other proteins. The amide I analysis here is a refinement of earlier work while the amide III analysis is new. Measurement of the amide I spectrum may be restricted to the region between 1500 and 1800 cm-1 in order to save time and increase the signal-to-noise ratio. The spectrum of buffer is subtracted from the amide III region to satisfy the criteria described for the amide I analysis. However, if the protein concentration is sufficiently high (2%) solvent correction may not be necessary, as the spectrum of water in this region is relatively linear. When samples are not particularly fluorescent, good results can be obtained by collecting the spectrum from 1100 to 1500 cm-1 and by fitting a straight line through the minima. The amide III spectrum is then 9-point smoothed using the algorithm by Savitsky and Golay.