The guanidine hydrochloride induced equilibrium denaturation of insulin and proinsulin was studied by using near- and far-ultraviolet (UV) circular dichroism (CD). The denaturation transition of insulin is reversible, cooperative, symmetrical, and the same whether detected by near- or far-UV CD. These results are consistent with a two-state denaturation process without any appreciable equilibrium intermediates. Analysis of the insulin denaturation data yields a Gibbs free energy of unfolding of 4.5 +/- 0.5 kcal/mol. Denaturation of proinsulin detected by near-UV CD appears to be the same as for insulin, but if detected by far-UV CD appears different. The far-UV CD results demonstrate a multiphasic transition with the connecting peptide portion unfolding at lower concentrations of denaturant. Similar studies with the isolated C-peptide show that its conformation and susceptibility to denaturation are independent of the rest of the proinsulin molecule. After the proinsulin denaturation results were adjusted for the connecting peptide contribution, a denaturation transition identical with that of insulin was obtained. These results show that for proinsulin, the connecting peptide segment is not a random coil; it is an autonomous folding unit, and the portion corresponding to insulin is identical with insulin in terms of conformational stability. Study holds ProTherm entries: 3650 Extra Details: additive : EDTA(1 mM), reversible; two-state denaturation;,free energies of unfolding; conformational stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:22 p.m.
|Number of data points||1|
|Proteins||Insulin ; Insulin|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence A:GIVEQCCTSICSLYQLENYCN/B:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/C:GIVEQCCTSICSLYQLENYCN/D:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/E:GIVEQCCTSICSLYQLENYCN/F:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/G:GIVEQCCTSICSLYQLENYCN/H:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/I:GIVEQCCTSICSLYQLENYCN/J:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/K:GIVEQCCTSICSLYQLENYCN/L:FVNQHLCGSHLVEALYLVCGERGFFYTPKT|