Estimation of protein secondary structure from circular dichroism spectra:Inclusion of denatured proteins with native proteins in the analysis

We have expanded our reference set of proteins used in the estimation of pr otein secondary structure by CD spectroscopy from 29 to 37 proteins by incl uding 3 additional globular proteins with known X-ray structure and 5 denat ured proteins. We have also modified the self-consistent method for analyzi ng protein CD spectra, SELCON3, by including a new selection criterion deve loped by W. C. Johnson, Jr. (Proteins Struct. Funct. Genet. 35, 307-312, 19 99). The secondary structure corresponding to the denatured proteins was ap proximated to be 90% unordered, owing to the spectral similarity of the den atured proteins and unordered structures. We examined the thermal denaturat ion of ribonuclease T1 by CD using both the original and expanded sets of r eference proteins and obtained more consistent results with the expanded se t. The expanded set of reference proteins will be helpful for the determina tion of protein secondary structure from protein CD spectra with higher rel iability, especially of proteins with significant unordered structure conte nt and/or in the course of denaturation. (C) 2000 Academic Press.