THE ROLE OF THE C-TERMINUS OF THE INSULIN B-CHAIN IN MODULATING STRUCTURAL AND FUNCTIONAL-PROPERTIES OF THE HORMONE

Within the scope of structure-function studies on the proteohormone in sulin, the role of the C-terminal segment B26-B30 for self-association and receptor interaction was analyzed. Insulin derivatives with modif ications in the region B26-B30 were synthesized by trypsin-catalyzed c oupling reactions of des-(B23-B30)-insulin with synthetic peptides. Th e peptides were obtained by Fmoc solid-phase peptide synthesis. Insuli ns with multiple amino acid --> glycine substitutions were examined to distinguish between the influence of the side chains and the influenc e of the main chain in positions B27-B30 on the self-association of th e hormone. The analogues [Gly(B27,B28,B29,B30)]insulin and [Gly(B27,B2 8,B30)]insulin exhibit relative receptor affinities of 80% and self-as sociate. The successive extension of [Ala(B26)]des-(B27-B30)-insulin-B 26-amide (relative receptor binding 273%) with amino acids correspondi ng to the native sequence B27-B30 showed the influence of the length o f the B-chain on receptor affinity: the extension by B27-threonine ami de reduces receptor binding to 71%, all further prolongations have onl y small effects on the binding. The effect of the B28-side chain on ma in-chain conformation, self-association and receptor binding was exami ned with [X(B28)]des-(B29-B30)-insulin-B28-amides(X = Phe, Gly, D-Pro) . While the glycine and D-proline analogues (relative binding 104 and 143%, respectively) retain the self-association properties typical of insulin, [Phe(B28)]des-(B29-B30)insulin-B28-amide(relative binding 50% ) shows diminished self-association. The backbone-modified insulin der ivative Sar(B26)]des-(B27-B30)-insulin-B26-amide(sarcosine = N-methylg lycine) exhibits an unexpectedly high receptor affinity of 1100% which demonstrates that the B26-amide hydrogen of the native hormone is not important for receptor binding. (C) Munksgaard 1995.