Quantification of protein secondary structure content by multivariate analysis of deep-ultraviolet resonance Raman and circular dichroism spectroscopies

Determination of protein secondary structure (alpha- helical, beta- sheet, and disordered motifs) has become an area of great importance in biochemistry and biophysics as protein secondary structure is directly related to protein function and protein related diseases. While NMR and X-ray crystallography can predict the placement of each atom in a protein to within an angstrom, optical methods (i.e. CD, Raman, and IR) are the preferred techniques for rapid evaluation of protein secondary structure content. Such techniques require calibration data to predict unknown protein secondary structure content where accuracy may be improved with the application of multivariate analysis. Here, a comparison of the protein secondary structure predictions obtained from multivariate analysis of ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopic data using classical least squares (CLS),partial least squares (PLS),and multivariate curve resolution-alternating least squares (MCR- ALS)is made. Results of the multivariate analysis suggest that CD measurements provide more accurate prediction of protein alpha-helical content whereas UVRR more accurately predicts beta-sheet content, an observation that is consistent with previous studies. Based on this analysis, it is suggested that the best approach to rapid and accurate protein secondary structure determination is to combine both CD and UVRR spectroscopic data.