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. Study holds ProTherm entries: 5157 Extra Details: additive : EDTA(1 mM), conformational stability; flexibility; H2O/D2O amide exchange;,hydrodynamic properties; receptor binding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:28 p.m.
|Number of data points||2|
|Proteins||Insulin ; Insulin|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence A:GIVEQCCTSICSLYQLENYCN/B:FVNQHLCGSHLVEALYLVCGERGFFYTPKA|