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Controlling the aggregation and rate of release in order to improve insulin formulation: molecular dynamics study of full-length insulin amyloid oligomer models

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Abstract

Insulin is a hormone that regulates the physiological glucose level in human blood. Insulin injections are used to treat diabetic patients. The amyloid aggregation of insulin may cause problems during the production, storage, and delivery of insulin formulations. Several modifications to the C-terminus of the B chain have been suggested in order to improve the insulin formulation. The central fragments of the A and B chains (LYQLENY and LVEALYL) have recently been identified as β-sheet-forming regions, and their microcrystalline structures have been used to build a high-resolution amyloid fibril model of insulin. Here we report on a molecular dynamics (MD) study of single-layer oligomers of the full-length insulin which aimed to identify the structural elements that are important for amyloid stability, and to suggest single glycine mutants in the β-sheet region that may improve the formulation. Structural stability, aggregation behavior and the thermodynamics of association were studied for the wild-type and mutant aggregates. A comparison of the oligomers of different sizes revealed that adding strands enhances the internal stability of the wild-type aggregates. We call this “dynamic cooperativity”. The secondary structure content and clustering analysis of the MD trajectories show that the largest aggregates retain the fibril conformation, while the monomers and dimers lose their conformations. The degree of structural similarity between the oligomers in the simulation and the fibril conformation is proposed as a possible explanation for the experimentally observed shortening of the nucleation lag phase of insulin with oligomer seeding. Decomposing the free energy into electrostatic, van der Waals and solvation components demonstrated that electrostatic interactions contribute unfavorably to the binding, while the van der Waals and especially solvation effects are favorable for it. A per-atom decomposition allowed us to identify the residues that contribute most to the binding free energy. Residues in the β-sheet regions of chains A and B were found to be the key residues as they provided the largest favorable contributions to single-layer association. The positive ∆∆G mut values of 37.3 to 1.4 kcal mol−1 of the mutants in the β-sheet region indicate that they have a lower tendency to aggregate than the wild type. The information obtained by identifying the parts of insulin molecules that are crucial to aggregate formation and stability can be used to design new analogs that can better control the blood glucose level. The results of our simulation may help in the rational design of new insulin analogs with a decreased propensity for self-association, thus avoiding injection amyloidosis. They may also be used to design new fast-acting and delayed-release insulin formulations.

Molecular dynamic study of the full length insulin amyloid oligomers identified structural elements important for their stability. Comparison of the aggregates of different size revealed that addition of strands enhances the internal stability of the oligomers. Per-atom decomposition of the binding free energy allowed us to identify the residues contributing most to the binding free energy. We found the residues in the β-sheet regions of chain A and chain B to be the key residues for the single layer association. The result from our simulation could help in the rational design of the new insulin analogues with the decreased propensity for self-association avoiding injection amyloidosis. It can also be used to design new fast acting and delayed release insulin formulations.

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Acknowledgments

This work was supported in part by the National Science Foundation (CCF/CHE 0832622). This research used resources of the National Energy Research Scientific Computing Center, which is supported by the Office of Science of the U.S. Department of Energy under contract no. DE-AC02-05CH11231.

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Authors and Affiliations

  1. NanoScience Technology Center and Department of Chemistry, University of Central Florida, Orlando, FL, 32826, USA

    Workalemahu Mikre Berhanu

  2. NanoScience Technology Center, Department of Chemistry, and Department of Physics, University of Central Florida, Orlando, FL, 32826, USA

    Artëm E. Masunov

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  1. Workalemahu Mikre Berhanu
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Correspondence to Artëm E. Masunov.

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Table 1S

Summary of the simulated single-layer insulin oligomer aggregate system (DOC 52 kb)

Table 2S

Clustering metric values for the wild-type clusters per system (DOC 62 kb)

Table 3S

Decomposition of the free energy on a per-residue basis for key residues in the monomer association of the single-layer ten-stranded insulin SH1-ST10. (A) Total Binding Free Energy Decomposition. (B) Decomposition of the contribution of the side chains to the binding free energy. (C) Decomposition of the contribution of the backbone to the binding free energy (DOC 110 kb)

Table 4S

Decomposition of the free energy on a per-residue basis for residues in chain A of the monomer association of the single-layer ten-stranded insulin SH1-ST10 and mutants of that insulin in chain A (DOC 150 kb)

Table 5S

Decomposition of the free energy on a per-residue basis for residues in chain A of the monomer association of the single-layer ten-stranded insulin SH1-ST10 and mutants of that insulin in chain B (DOC 251 kb)

Fig. S1

Secondary structural elements of the insulin model (initial structure). Helices are shown as magenta ribbons, β-strands as yellow arrows, and the rest are shown as loops. The positions of the disulfide bonds are indicated by a blue line (JPEG 110 kb)

High-resolution image (TIFF 455 kb)

Fig. S2

Superposition of the initial structures of single-layer insulin oligomer aggregates on the most representative structures of the most populated clusters (A SH1-ST1, B SH1-ST2, C SH1-ST4, D SH1-ST6, E SH1-ST4, and F SH1-ST6) for the corresponding aggregates. The initial structures are shown in blue and the most populated clusters with their corresponding cluster occupancies (in %) are shown in magenta (JPEG 127 kb)

High-resolution image (TIFF 346 kb)

Fig. S3

Profile of ∆∆G against the number of chains in single-layer insulin oligomer aggregate nucleation fibrillation. When the number of chains is high, the oligomer is stable and has favorable free energy (JPEG 44 kb)

High-resolution image (TIFF 55 kb)

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Berhanu, W.M., Masunov, A.E. Controlling the aggregation and rate of release in order to improve insulin formulation: molecular dynamics study of full-length insulin amyloid oligomer models. J Mol Model 18, 1129–1142 (2012). https://doi.org/10.1007/s00894-011-1123-3

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