Statistical analyses of the vibrational circular dichroism of selected proteins and relationship to secondary structures.

The vibrational circular dichroism (VCD) spectra of 20 proteins dissolved in D2O are presented in the amide I' region. These data are decomposed into a linear combination of orthogonal subspectra generated by the principal component method of factor analysis, and the results for 13 of them are compared to their secondary structures as determined from X-ray crystallography. Factor analysis of the VCD yields six statistically significant subspectra that can be used to reproduce the spectra. Their coefficients can then be used to characterize a given protein. Comparison of cluster analyses of these VCD coefficients and of the secondary structure fractional coefficients from X-ray crystallography showed that proteins clustered in the VCD analysis were also clustered in the X-ray analysis. The relative fractions of alpha-helix and beta-sheet in the protein dominate the clustering in both data sets. Qualitative characterization of the secondary structure of a given protein is obtained from its clustering on the basis of spectral characteristics. A strong linear correlation was found between the coefficient of the second subspectrum and the alpha-helical fraction for the proteins studied. The second coefficient also correlated to the beta-sheet fraction, and the first coefficient weakly correlated to the fraction for "other". Subsequent multiple-parameter regression analyses of the VCD factor analysis coefficients, constrained to include only significant dependencies, yielded reliable determination of the alpha-helix fraction and somewhat less confident determination of beta-sheet, bend, and "other" components. Predictive capability for proteins not in the regression was good. Varimax rotation of the coefficients transformed the subspectra and gave simple correlations to secondary structure components but had less reliability and more restrictions than the multiple regression on the original coefficients. The partial least-squares analysis method was also used to predict fractional secondary structures for the training set proteins but resulted in somewhat higher average error, particularly for beta-sheet, than the multiple regression. The turn fraction was effectively undetermined in both the regression and partial least-squares analyses. These statistical analyses represent the first determination of a quantitative relationship between VCD spectra and secondary structure in proteins.

Conformational transitions in phosvitin with pH variation. Vibrational circular dichroism study.

The vibrational circular dichroism (VCD) spectra of metal-free phosvitin are presented as a function of pH and analyzed both qualitatively and by using a factor analysis approach referenced to a protein data set. The qualitative pattern of both the IR and VCD changes is consistent with a coil-to-sheet transition occurring as pH is progressively decreased to values lower than 3. A similar transition was seen in commercial preparation of phosvitin which still contained metal ions, but there the transition was more gradual and occurred at somewhat different pH values. Such a gradual change is also evident in the solution phase absorption band profile but is made clearer using Fourier deconvolution. Based on VCD results, the low pH transition appears to occur with two distinct manifestations of the beta-sheet form. However, at the lowest pH values the sample may precipitate. These two forms are not distinguishable with Fourier transform infrared alone and may be due to a twist of the beta-sheet form or to aggregation.

Enhanced sensitivity to conformation in various proteins. Vibrational circular dichroism results.

Vibrational circular dichroism (VCD) spectra of several globular proteins dissolved in D2O are presented and compared to conventional UV-CD results. It can be seen that, for the alpha, beta, and alpha + beta categories of Levitt and Chothia [(1976) Nature 261, 552], VCD evidences much larger band shape variations, including sign alteration, than does UV-CD. A direct parallel is seen between the VCD of the alpha-helix found in model polypeptides and the amide I' VCD of myoglobin. Since all structural aspects of the protein contribute to the VCD on a roughly equal footing, a similar correlation of the chymotrypsin amide I' VCD with that of beta-sheet models is not as clear. In addition, the VCD of "random-coil"-type proteins is found to be clearly related to VCD results from "random-coil" polypeptides. Finally, simulations are presented to postulate the expected VCD for protein structures having conformations that lie between the limiting cases discussed here.