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Biophys J. 2019 Nov 5;117(9):1626-1641. doi: 10.1016/j.bpj.2019.09.022. Epub 2019 Sep 23.

Modulating Insulin Fibrillation Using Engineered B-Chains with Mutated C-Termini.

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Protein Chemistry Laboratory, Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran.
Protein Chemistry Laboratory, Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran. Electronic address:
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Department of Biology, East Carolina University, Greenville, North Carolina.
Department of Molecular Medicine and Byrd Alzheimer's Center and Research Institute, Morsani College of Medicine, University of South Florida, Tampa, Florida; Laboratory of New Methods in Biology, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Oblast, Russia.


Stress-induced unfolding and fibrillation of insulin represent serious medical and biotechnological problems. Despite many attempts to elucidate the molecular mechanisms of insulin fibrillation, there is no general agreement on how this process takes place. Several previous studies suggested the importance of the C-terminal region of B-chain in this pathway. Therefore, we generated the T30R and K29R/T30R mutants of insulin B-chain. Recombinantly produced wild-type A-chain and mutant B-chains were combined efficiently in the presence of chaperone αB-crystallin. The mutant B-chains along with the control wild-type insulin were used in a wide range of parallel experiments to compare their fibrillation kinetics, morphology of fibrils, and forces driving the fibril formation. The mutant insulins and their B-chains displayed significant resistance against stress-induced fibrillation, particularly at the nucleation stage, suggesting that the B-chain might be influencing the insulin fibrillation. The fact that the different mature insulins formed larger fibrillar bundles compared to those formed by their B-chains alone suggested the role of A-chain in the lateral association of the insulin fibrils. Overall, in addition to the N-terminal region of the B-chain, which was shown to serve as an important regulator of insulin fibrillation, the C-terminal region of this peptide is also crucial for the control of fibrillation, likely serving as an attachment site engaged in the formation of the nucleus and protofibril. Finally, two mutated insulin variants examined in this study might be of interest to the pharmaceutical sector as, to our knowledge, novel intermediate-acting insulin analogs because of their suitable biological activity and improved stability against stress-induced fibrillation.

[Available on 2020-11-05]

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