Conflict of interest: All authors employed at Novo Nordisk own Novo Nordisk stocks.
Insulin analog with additional disulfide bond has increased stability and preserved activity†
Article first published online: 17 JAN 2013
Copyright © 2012 The Protein Society
Volume 22, Issue 3, pages 296–305, March 2013
How to Cite
Vinther, T. N., Norrman, M., Ribel, U., Huus, K., Schlein, M., Steensgaard, D. B., Pedersen, T. Å., Pettersson, I., Ludvigsen, S., Kjeldsen, T., Jensen, K. J. and Hubálek, F. (2013), Insulin analog with additional disulfide bond has increased stability and preserved activity. Protein Science, 22: 296–305. doi: 10.1002/pro.2211
- Issue published online: 18 FEB 2013
- Article first published online: 17 JAN 2013
- Accepted manuscript online: 26 DEC 2012 05:30AM EST
- Manuscript Accepted: 7 DEC 2012
- Manuscript Revised: 4 DEC 2012
- Manuscript Received: 4 OCT 2012
- Danish Ministry of Science, Technology and Innovation and the Novo Nordisk STAR program
- protein design;
Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.