Molecular Details of a Salt Bridge and Its Role in Insulin Fibrillation by NMR and Raman Spectroscopic Analysis

March 24, 2020

Title

Molecular Details of a Salt Bridge and Its Role in Insulin Fibrillation by NMR and Raman Spectroscopic Analysis

Author

Bhisma N. Ratha, Rajiv K. Kar, Zuzana Bednarikova, Zuzana Gazova, Samuel A. Kotler, Sreyan Raha, Soumya De, Nakul C. Maiti, Anirban Bhunia

Year

2020

Journal

Journal of Physical Chemistry B

Abstract

Insulin, a simple polypeptide hormone with huge biological importance, has long been known to self-assemble in vitro and form amyloid-like fibrillar aggregates. Utilizing high-resolution NMR, Raman spectroscopy, and computational analysis, we demonstrate that the fluctuation of the carboxyl terminal (C-ter) residues of the insulin B-chain plays a key role in the growth phase of insulin aggregation. By comparing the insulin sourced from bovine, human, and the modified glargine (GI), we observed reduced aggregation propensity in the GI variant, resulting from two additional Arg residues at its C-ter. NMR analysis showed atomic contacts and residue-specific interactions, particularly the salt bridge and H-bond formed among the C-ter residues Arg31B, Lys29B, and Glu4A. These inter-residue interactions were reflected in strong nuclear Overhauser effects among Arg31BδH–Glu4AδH and Lys29BδHs–Glu4AδH in GI, as well as the associated downfield chemical shift of several A-chain amino terminal (N-ter) residues. The two additional Arg residues of GI, Arg31B and Arg32B, enhanced the stability of the GI native structure by strengthening the Arg31B, Lys29B, and Glu4A salt bridge, thus reducing extensive thermal distortion and fluctuation of the terminal residues. The high stability of the salt bridge retards tertiary collapse, a crucial biochemical event for oligomerization and subsequent fibril formation. Circular dichroism and Raman spectroscopic measurement also suggest slow structural distortion in the early phase of the aggregation of GI because of the restricted mobility of the C-ter residues as explained by NMR. In addition, the structural and dynamic parameters derived from molecular dynamics simulations of insulin variants highlight the role of residue-specific contacts in aggregation and amyloid-like fibril formation.

Instrument

J-815

Keywords

Circular dichroism, Secondary structure, Aggregation, Kinetics, Biochemistry