The conformational stability and flexibility of insulin with an additional intramolecular cross-link.
The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.
Brems
D N
DN
Department of Parenteral Research and Development, Eli Lilly & Co., Indianapolis, Indiana 46285.
Brown
P L
PL
Nakagawa
S H
SH
Tager
H S
HS
D000818:Animals; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003903:Deuterium / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D066298:In Vitro Techniques; D007328:Insulin / Q000737:chemistry; D009038:Motion; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D013552:Swine
D003432:Cross-Linking Reagents; D006146:Guanidines; D007328:Insulin; D003903:Deuterium; D019791:Guanidine