Additional disulfide bonds in insulin: Prediction, recombinant expression, receptor binding affinity, and stability

作者：Vinther Tine N^*; Pettersson Ingrid; Huus Kasper; Schlein Morten; Steensgaard Dorte B; Sorensen Anders; Jensen Knud J; Kjeldsen Thomas; Hubalek Frantisek

来源：Protein Science, 2015, 24(5): 779-788.

DOI：10.1002/pro.2649

• disulfide
• prediction
• insulin
• protein design
• stability

The structure of insulin, a glucose homeostasis-controlling hormone, is highly conserved in all vertebrates and stabilized by three disulfide bonds. Recently, we designed a novel insulin analogue containing a fourth disulfide bond located between positions A10-B4. The N-terminus of insulin's B-chain is flexible and can adapt multiple conformations. We examined how well disulfide bond predictions algorithms could identify disulfide bonds in this region of insulin. In order to identify stable insulin analogues with additional disulfide bonds, which could be expressed, the C cut-off distance had to be increased in many instances and single X-ray structures as well as structures from MD simulations had to be used. The analogues that were identified by the algorithm without extensive adjustments of the prediction parameters were more thermally stable as assessed by DSC and CD and expressed in higher yields in comparison to analogues with additional disulfide bonds that were more difficult to predict. In contrast, addition of the fourth disulfide bond rendered all analogues resistant to fibrillation under stress conditions and all stable analogues bound to the insulin receptor with picomolar affinities. Thus activity and fibrillation propensity did not correlate with the results from the prediction algorithm.

• 出版日期2015-5