Protein crystallization: universal thermodynamic vs. specific effects of PEG

Abstract

The interest of nucleation of protein crystals and aggregates (including oligomerization) spans from basic physics theory all the way to biophysics, nanophysics, clinical sciences, biotechnologies, food technologies and polymer-solvent interactions. Understanding nucleation within a theoretical framework capable of providing quantitative predictions and control of nucleation rates, or even the very occurrence of crystallization, is a long-sought goal of remarkable relevance to each of the above fields. A large amount of work has been aimed at such goal, but success has been so far rather limited. Work at our laboratory has more recently highlighted a direct link between nucleation rates and the universal scaling properties of concentration fluctuations occurring in the vicinity of a phase transition. The phase transition here concerned is that of non nucleated liquid-liquid demixing of the solution. This novel universality feature allows viewing nucleation processes within one and the same frame, and to capture all normalized nucleation rates on one and the same "master curve" for different proteins, as a function of one parameter only. The quantitative value of the latter is the result of the joint, non additive effects of protein composition, conformation and state (e.g. oligomers), as well as of the temperature of non nucleated liquid-liquid demixing of the solution at the given protein concentration and at the given conditions of the solution. The present work was undertaken for the purpose of ascertaining if (and if so, in what way) the universality feature can allow the quantitative prediction of nucleation rates changes caused by the addition to the solvent of additives empirically known for their strong effect on such rates, as well as the very occurrence of crystallization. To this purpose we have used PEG (polyethylene glycol), which is perhaps the most familiar and most-used additive, and have measured by static and dynamic light scattering the properties of concentration fluctuation of the system as a function of temperature, for various PEG concentration and polymerisation degrees. Experiments have shown that the action of PEG can in no way be accounted for in terms of changes of specific local contacts or of a one-to-one chaperone-like action. Instead, the effect of PEG is seen to be due to alteration of the thermodynamic properties of the solution. This leaves unchanged the universality features and consequently also the validity and predictive power of the master curve in the various conditions.