Primary Steps of pH‐Dependent Insulin Aggregation Kinetics are Governed by Conformational Flexibility

Abstract

Insulin aggregation critically depends on pH. The underlying energetic and structural determinants are, however, unknown. Here, we measure the kinetics of the primary aggregation steps of the insulin monomer in vitro and relate it to its conformational flexibility. To assess these primary steps the monomer concentration was monitored by mass spectrometry at various pH values and aggregation products were imaged by atomic force microscopy. Lowering the pH from 3 to 1.6 markedly accelerated the observed aggregation kinetics. The influence of pH on the monomer structure and dynamics in solution was studied by molecular dynamics simulations, with the protonation states of the titrable groups obtained from electrostatic calculations. Reduced flexibility was observed for low pH values, mainly in the C terminus and in the helix of the B chain; these corresponded to an estimated entropy loss of 150 J mol(-1) K(-1). The striking correlation between entropy loss and pH value is consistent with the observed kinetic traces. In analogy to the well-known Phi value analysis, this result allows the extraction of structural information about the rate determining transition state of the primary aggregation steps. In particular, we suggest that the residues in the helix of the B chain are involved in this transition state.

Constant pH Molecular Dynamics in Explicit Solvent with λ-Dynamics

“…Most notably, many proteins denature at low pH values,(1) and aggregation, such as formation of amyloid fibrils in Alzheimer’s disease(2) and insulin aggregation,(3) is pH-dependent. Because the function of a protein depends on its structure, pH is critical for protein function.…”

Spontaneous Aggregation of the Insulin-Derived Steric Zipper Peptide VEALYL Results in Different Aggregation Forms with Common Features

“…The recently resolved three-dimensional crystal structure of VEALYL reveals a class 7 zipper, characterized by a face-to-back packing of the β-sheets, and antiparallel stacked strands within the sheets [18]. The VEALYL segment is proposed to play an important role in full-length insulin misfolding [34,35,18,36] and to participate in the β-strand core region of mature insulin fibrils [37,38].…”

The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG

“…The formation of insulin amyloid fibrils in vivo is associated with the clinical syndrome injection-localised amyloidosis, which was observed in diabetes patients after continuous subcutaneous injections of insulin [50,51]. Despite its main application in medicine, recombinant human insulin is also extensively used as a model protein to study pathways and mechanisms of amyloid fibril formation in vitro.…”