Conformational analysis of circular dichroism spectra of insulin, proinsulin and c-peptides by non-linear regression.

A method of resolving CD spectra in alpha-helix, beta-structure and random coil conformations is described. The residue ellipticites for alpha-helix and beta-structure given by Greenfield & Fasman or by Chen,, Yang & Martinez are used together with CD spectra from at least two similar peptides to determine, by an iterative least-squares method, the number of amino acids in the three reference conformations as well as a set of residue ellipticities characteristic of the random coils of the family of peptides in question, but not necessarily of other peptides. The fits between computed and experimental spectra improve significantly and systematic deviations disappear by allowing the random coil coefficients to vary from one family of proteins to another, a liberty justified by the different types of random coils that have been encountered. The method of analysis showed that 5 M urea did not change the conformations of C-peptides of proinsulin from ox, pig and duck, all being mainly in the random coil conformation and all having 3-4 amino acids in beta-structure. Bovine insulin and proinsulin showed a transfer of amino acids from alpha-helix to beta-structure with increasing concentrations of urea, the latter at a higher concentration, indicating a stabilizing effect of the connecting peptide. The numbers of amino acids found in the alpha-helical conformation in insulin and proinsulin were equal and in agreement with the X-ray crystallographic data for insulin when the Greenfield & Fasman coefficients for alpha-helix and beta-structure were employed, whereas the Chen, Yang & Martinez coefficients yielded too few amino acids in alpha-helix in proinsulin. Both sets of coefficients estimate more beta-structure in proinsulin than in insulin.