Improved estimation of the secondary structures of proteins by vacuum-ultraviolet circular dichroism spectroscopy

The vacuum-ultraviolet circular dichroism (VUVCD) spectra of 16 globular proteins (insulin, lactate dehydrogenase, glucose isomerase, lipase, conalbumin, transferrin, catalase, subtilisin A, a-amylase, staphylococcal nuclease, papain, thioredoxin, carbonic anhydrase, elastase, avidin, and xylanase) were successfully measured in aqueous solutions at 25 degrees C from 260 to 160 run under a high vacuum using a synchrotronradiation VUVCD spectrophotometer. These proteins exhibited characteristic CD spectra below 190 nm that were related to their different secondary structures, which could not be detected with a conventional CD spectrophotometer. The component spectra of a-helices, P-strands, turns, and unordered structures were obtained by deconvolution analysis of the VUVCD spectra of 31 reference proteins including the 15 proteins reported in our previous paper [Matsuo, K. et aL (2004) J. Biochem. 135, 405-411]. Prediction of the secondary-structure contents using the SELCON3 program was greatly improved, especially for a-helices, by extending the short-wavelength limit of CD spectra to 160 nm and by increasing the number of reference proteins. The numbers of a-helix and P-strand segments, which were calculated from the distorted a-helix and P-strand contents, were close to those obtained on X-ray crystallography. These results demonstrate the usefulness of synchrotron-radiation VUVCD spectroscopy for the secondary structure analysis of proteins.