Time scale of protein aggregation dictated by liquid‐liquid demixing

Abstract

The growing impact of protein aggregation pathologies, together with the current high need for extensive information on protein structures are focusing much interest on the physics underlying the nucleation and growth of protein aggregates and crystals. Sickle Cell Hemoglobin (HbS), a point-mutant form of normal human Hemoglobin (HbA), is the first recognized and best-studied case of pathologically aggregating protein. Here we reanalyze kinetic data on nucleation of deoxy-HbS aggregates by referring them to the (concentration-dependent) temperature T(s) characterizing the occurrence of the phase transition of liquid-liquid demixing (LLD) of the solution. In this way, and by appropriate scaling of kinetic data at different concentrations, so as to normalize their spans, the apparently disparate sets of data are seen to fall on a master curve. Expressing the master curve vs. the parameter epsilon = (T - T(s)) / T(s), familiar from phase transition theory, allows eliciting the role of anomalously large concentration fluctuations associated with the LLD phase transition and also allows decoupling quantitatively the role of such fluctuations from that of microscopic, inter-protein interactions leading to nucleation. Referring to epsilon shows how in a narrow temperature span, that is at T - T(s), nucleation kinetics can undergo orders-of-magnitude changes, unexpected in terms of ordinary chemical kinetics. The same is true for similarly small changes of other parameters (pH, salts, precipitants), capable of altering T(s) and consequently epsilon. This offers the rationale for understanding how apparently minor changes of parameters can dramatically affect protein aggregation and related diseases.

Lysozyme crystallization rates controlled by anomalous fluctuations

“…1 and 3 left, one sees that close to, or past the spinodal line, amorphous precipitation occurs. This is at variance with the case of HbS, where nucleation data extend well inside the demixing region, still falling on the continuation of the same master curve [44] (the panel related to the actual demixing region is omitted from Fig. 3).…”

“…In the same figure, we also show a pertinent part of recent results concerning sickle hemoglobin (HbS) fiber formation, obtained by a similar approach [44]. In that case too, classic literature data on induction times for different HbS concentrations and temperatures were used, appropriately rescaled and referred to T S and as in the present case.…”

“…This offers a deeper and quantitative insight into the effect of parameter changes [39][40][41][42] and the possibility of compensating on a basis of quantitative knowledge, for the change of some parameters by the change of others. Finally, referring to allows putting on quantitative grounds the long proposed approach to aggregation/crystallization control [15,[39][40][41][42]44,45,51,52].…”

“…For this reason, a concentration-dependent normalization factor is not required for lysozyme, while it is necessary for the HbS case [44,45]. Best fit of experimental points relative to both lysozyme and HbS to Eq.…”

Systematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants

“…A major reason is that the mutations A4V and V148G yield aggregation temperatures that are much higher than anticipated from their thermodynamic and chemical properties alone. One possibility is that these highly destabilized mutants aggregate by alternative pathways involving an early decomposition of the starting material into small precursor aggregates (47,48). Such local decomposition does not need to show up in the light-scattering experiments and could contribute to underestimate the intrinsic stickiness of the free monomers.…”