Mellitate: A multivalent anion with extreme charge density causes rapid aggregation and misfolding of wild type lysozyme at neutral pH

Abstract

Due to its symmetric structure and abundance of carboxyl groups, mellitic acid (MA–benzenehexacarboxylic acid) has an uncommon capacity to form highly ordered molecular networks. Dissolved in water, MA dissociates to yield various mellitate anions with pronounced tendencies to form complexes with cations including protonated amines. Deprotonation of MA at physiological pH produces anions with high charge densities (MA5- and MA6-) whose influence on co-dissolved proteins has not been thoroughly studied. As electrostatic attraction between highly symmetric MA6- anions and positively charged low-symmetry globular proteins could lead to interesting self-assembly patterns we have chosen hen egg white lysozyme (HEWL), a basic stably folded globular protein as a cationic partner for mellitate anions to form such hypothetical nanostructures. Indeed, mixing of neutral HEWL and MA solutions does result in precipitation of electrostatic complexes with the stoichiometry dependent on pH. We have studied the self-assembly of HEWL-MA structures using vibrational spectroscopy (infrared absorption and Raman scattering), circular dichroism (CD), atomic force microscopy (AFM). Possible HEWL-MA6- molecular docking scenarios were analyzed using computational tools. Our results indicate that even at equimolar ratios (in respect to HEWL), MA5- and MA6- anions are capable of inducing misfolding and aggregation of the protein upon mild heating which results in non-native intermolecular beta-sheet appearing in the amide I’ region of the corresponding infrared spectra. The association process leads to aggregates with compacted morphologies entrapping mellitate anions. The capacity of extremely diluted mellitate anions (i.e. at sub-millimolar concentration range) to trigger aggregation of proteins is discussed in the context of mechanisms of misfolding.

Controlling Phase Separation of Lysozyme with Polyvalent Anions

“…50 Mellitate, a small multivalent anion, precipitates lysozyme at very low salt concentrations, but no resolubilisation occurs at higher salt concentration. 51 As such, it is not clear to what extent the universal behaviour observed for trivalent metal cations with negatively charged proteins can be transferred to multivalent anions interacting with positively charged proteins.…”

“…The multivalent cation spermidine is not capable of causing precipitation of negatively charged proteins, which has been attributed to the diffuse charge distribution on the polycation . Mellitate, a small multivalent anion, precipitates lysozyme at very low salt concentrations, but no resolubilization occurs at higher salt concentration . As such, it is not clear to what extent the universal behavior observed for trivalent metal cations with negatively charged proteins can be transferred to multivalent anions interacting with positively charged proteins.…”

Model for Counterion Binding and Charge Reversal on Protein Surfaces

“…Buffers that are added to maintain the pH of protein solutions also interact with protein surfaces, , alter the surface charge, and consequently affect protein stability . Polyvalent anions and cations at varying ionic strengths have been shown to strongly affect net protein charge, to the extent of protein surface charge reversal in some cases. ,− Since protein stability is sensitive to net charge, tuning of protein complexation with polyvalent ions could assist in the prevention of aggregation. A recent report demonstrates that phase separation of lysozyme, at a pH where it is overall positively charged, can be controlled with polyvalent anions, and relates this to the net charge carried by the lysozyme–polyvalent ion complexes, which includes protein charge reversal .…”