A kinetic coupling between protein unfolding and aggregation controls time‐dependent solubility of the human myeloma antibody light chain

Abstract

Protein aggregation is one of the most critical processes affecting protein solubility in various contexts-from protein therapeutics formulation to protein diseases. In general, time-dependent changes in protein solubility are complex kinetically driven processes that often involve a triggering event that consists of a protein unfolding/misfolding followed by the assembling of aggregationcompetent protein species. In this study, we have examined the relation between stability and time-dependent solubility of the recombinant human antibody light chain, hLC, which was found to form renal tubular casts in the multiple myeloma patient. To analyze the aggregation quantitatively, the hLC stability and protein solubility assays were performed in vitro at elevated temperatures. A differential acceleration of the processes at high temperatures enabled us to dissect observed kinetics of irreversible hLC unfolding and aggregation. We find that for hLC these processes have different molecularity and activation energy barriers. While the irreversible unfolding of hLC is a unimolecular step with a substantial activation energy barrier of 260 kJ/mol, the aggregation is rate-limited by the bimolecular reaction, which is characterized by a lower activation energy barrier of 40 kJ/mol. By the combination of experimental assays at different temperatures, different protein concentrations and kinetic modeling using ordinary differential equations, we were able to extrapolate time-dependent protein solubility to temperatures where both unfolding and aggregation processes are strongly kinetically coupled. Our study enables mechanism-based evaluation and interpretation of different physico-chemical factors contributing to the hLC unfolding and aggregation and their effect on the formation of extracellular protein deposits.

Aggregation mechanism and branched 3D morphologies of pathological human light chain proteins under reducing conditions

“…However, such slow dissociation would result in overall rate-limiting fibril growth, which has to be first-order concentration-independent, which was not observed. A piece of evidence that dimeric species can be used as a nucleus originates from our temperature-dependence study, where we observed large aggregates with disulfide bridges under oxidizing conditions [28]. We conclude that the nucleation may start equally from dimeric and monomeric species.…”

“…The sequence of human light chain (hLC) JTO was synthesized by Genscript, and protein was produced by a bacterial expression system and purified, as described previously [28]. The measurements were performed in 1x PBS buffer (pH 7.4).…”

“…Our previous study described a kinetic coupling between colloidal and conformational stability of human light chain protein (hLC) from the myeloma patient in detail [28]. In our previous study, we showed that light chains could form large aggregates upon heating.…”