Circular dichroic analysis of denatured proteins: inclusion of denatured proteins in the reference set.

We hypothesize that inclusion of denatured proteins in the set of reference native proteins may better represent the unordered form in the current circular dichroism (CD) analyses of proteins involving unfolding ones. Adding three denatured-protein spectra and one oligopeptide spectrum to 16 reference protein spectra markedly improved the correlation coefficients (r) between CD calculations and X-ray determinations for the unordered form and, to a lesser extent, for beta-turn, but the r-values for alpha-helix and beta-sheet decreased slightly. With 20 reference proteins the estimates of the unordered form of denatured proteins were significantly improved. Thus, we suggest that as a compromise the new set of reference proteins be used for estimating the changes in conformation for unfolding proteins. However, the current use of 16 reference native proteins appears to be adequate for CD analysis of native proteins and the expansion to 20 reference proteins including denatured ones may not enhance the analysis of native proteins.

Some problems of CD analyses of protein conformation.

The circular dichroic (CD) spectra of 16 reference proteins were analyzed by the Provencher-Glöckner method (Biochemistry 20, 31, 1981) with the lower-wavelength limit raised from 190 to 235 nm at 5-nm intervals. Fifty-one data points at 1-nm intervals were taken between 190 and 240 nm. Variations of the correlation coefficients (r) and root-mean-square (RMS) deviations between X-ray diffraction results and CD analyses showed no definite trend with shorter wavelength ranges. The CD spectra (190-240 nm) were also analyzed by assigning the secondary structure of X-ray results according to the Levitt-Greer method (J. Mol. Biol. 114, 181, 1977) and the Kabsch-Sander method (Biopolymers 22, 2577, 1983). The r and RMS values based on the Levitt-Greer assignment were good and comparable to those based on the secondary structure given by crystallographers, but the Kabsch-Sander assignment seemed to give unsatisfactory results. The choice of reference proteins remains one of the uncertainties in the CD analysis. The five most significant orthogonal spectra (190-240 nm) calculated from the 16 reference proteins and those based on another 16 proteins used by Hennessey and Johnson (Biochemistry 20, 1085, 1981) were similar to each other, but different in intensities. These methods still cannot recognize a failed analysis of unknown proteins without X-ray results to check their reliability.

Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. III. Estimation of the protein secondary structure.

Infrared spectra of 13 globular proteins have been obtained in the 1800-1480-cm-1 region for H2O solutions. A method for estimating protein secondary structure from the ir spectrum has been developed. The method can also be used for estimating polypeptide and fibrous protein conformation. For the globular and fibrous proteins and polypeptides analyzed, the correlation coefficients between the ir and x-ray estimates of ordered helix, disordered helix, ordered beta-structure, disordered beta-structure, turns, and remainder were 0.98, 0.80, 0.99, 0.87, 0.90, and 0.92 respectively.