Abstract
Biomolecular assembly processes based on liquid–liquid phase separation (LLPS) are ubiquitous in the biological cell. To fully understand the role of LLPS in biological self-assembly, it is necessary to characterize also their kinetics of formation and dissolution. Here, we introduce the pressure-jump relaxation technique in concert with UV/Vis and FTIR spectroscopy as well as light microscopy to characterize the evolution of LLPS formation and dissolution in a time-dependent manner. As a model system undergoing LLPS we used the globular eye-lens protein γD-crystallin. As cosolutes and macromolecular crowding are known to affect the stability and dynamics of biomolecular condensates in cellulo, we extended our kinetic study by addressing also the impact of urea, the deep-sea osmolyte trimethylamine-N-oxide (TMAO) and a crowding agent on the transformation kinetics of the LLPS system. As a prerequisite for the kinetic studies, the phase diagram of γD-crystallin at the different solution conditions also had to be determined. The formation of the droplet phase was found to be a very rapid process and can be switched on and off on the 1–4 s timescale. Theoretical treatment using the Johnson–Mehl–Avrami–Kolmogorov model indicates that the LLPS proceeds via a diffusion-limited nucleation and growth mechanism at subcritical protein concentrations, a scenario which is also expected to prevail within biologically relevant crowded systems. Compared to the marked effect the cosolutes take on the stability of the LLPS region, their effect at biologically relevant concentrations on the phase transformation kinetics is very small, which might be a particular advantage in the cellular context, as a fast switching capability of the transition should not be compromised by the presence of cellular cosolutes.
Highlights
Upon formation of the homogeneous phase, i.e. upon dissolution of the protein droplets, the kinetics is about a factor of 10 faster and similar for all solution conditions
Our experiments demonstrate that the deep-sea cosolute TMAO, an osmolyte upregulated in deep-sea fish, significantly enhances the stability of the condensed protein droplets, but does not significantly affect the rapid kinetics of LLPS formation and dissolution of globular protein condensates
Turbidity is linked to the Hessian matrix of second derivatives of the Gibbs free energy of the solution per unit volume with respect to the number densities of the components, which determines the stability of the solution with respect to phase separation[46]
Summary
Owing to the repulsive excluded volume effect imposed by the crowding agent, condensed structures are favored in such situation as well, which results in an increase of the temperature and pressure stability of the droplet phase of the protein. As the pressure dependent processes were fully reversible, bidirectional p-jumps could be carried out, which allowed us to determine the rates of formation and dissolution of the protein droplet phase.
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