Equilibrium Ensembles for Insulin Folding from Bias-Exchange Metadynamics

Abstract

Earliest events in the aggregation process, such as single molecule reconfiguration, are extremely important and the most difficult to characterize in experiments. To this end, we have used well-tempered bias exchange metadynamics simulations to determine the equilibrium ensembles of an insulin molecule under amyloidogenic conditions of low pH and high temperature. A bin-based clustering method that uses statistics accumulated in bias exchange metadynamics trajectories was employed to construct a detailed thermodynamic and kinetic model of insulin folding. The highest lifetime, lowest free-energy ensemble identified consisted of native conformations adopted by a folded insulin monomer in solution, namely, the R-, the R-, and the T-states of insulin. The lowest free-energy structure had a root mean square deviation of only 0.15 nm from native x-ray structure. The second longest-lived metastable state was an unfolded, compact monomer with little similarity to the native structure. We have identified three additional long-lived, metastable states from the bin-based model. We then carried out an exhaustive structural characterization of metastable states on the basis of tertiary contact maps and per-residue accessible surface areas. We have also determined the lowest free-energy path between two longest-lived metastable states and confirm earlier findings of non-two-state folding for insulin through a folding intermediate. The ensemble containing the monomeric intermediate retained 58% of native hydrophobic contacts, however, accompanied by a complete loss of native secondary structure. We have discussed the relative importance of nativelike versus nonnative tertiary contacts for the folding transition. We also provide a simple measure to determine the importance of an individual residue for folding transition. Finally, we have compared and contrasted this intermediate with experimental data obtained in spectroscopic, crystallographic, and calorimetric measurements during early stages of insulin aggregation. We have also determined stability of monomeric insulin by incubation at a very low concentration to isolate protein-protein interaction effects.

Misfolded proinsulin in the endoplasmic reticulum during development of beta cell failure in diabetes

“…10 Distinct crystallographic structures known as R and T states reflect dynamic differences in the amino terminal portion of the B-chain, which can be mostly ␣-helical (R state) or extended (T state). 11 Like many globular proteins, insulin has a number of buried hydrophobic residues in the native state that may become solvent exposed in incompletely folded states. 12 Such improperly exposed peptide sequences, especially those including the central B-chain helix LVEALYL, are thought to be aggregation prone/amyloidogenic 13 and can associate with neighboring insulin moieties, even when they are in the native state.…”

Insulin dissociates by diverse mechanisms of coupled unfolding and unbinding

“…However, the monomeric region has more variability in detachment angle than in the dimeric region, consistent with previous results showing a limited amount of disorder in the C-terminal segment of the B chain. [22][23][24]26 Along the β path, instead of unfolding, the β sheets separate and the monomers drift away from one another, forming a diverse set of non-specific, nonnative interfacial contacts, as in structure 2β in Figure 3. The numbers of native and nonnative interfacial contacts are plotted in the center and right panels of Figure 6C, respectively.…”

“…Specifically, experimental and computational studies indicate that Phe B24 -Ala B30 can detach from the B-chain α helix and become at least partially disordered in the monomeric state. 19,[21][22][23][24][25][26] This detachment is thought to be important for insulin to bind its receptor 3,12,13 based on structures of insulin in complex with fragments of the receptor. 27,28 An outstanding question is how Phe B24 -Ala B30 detachment is coupled to dissociation of the dimer.…”

Exploring high‐dimensional free energy landscapes of chemical reactions

“…A number of studies used BEMetaD to study protein folding . Literature of BEMetaD also includes conformational sampling in α ‐helical glycoproteins, Bovine Chymosin, peptides, conformational sampling of intrinsically disordered as well as other proteins, conduction through ion channels, and protein aggregation . The method has also helped to resolve the structure of large and complex systems such as protein–RNA complex, and membrane inserted influenza fusion peptide .…”