Chaperone salts, polyethylene glycol and rates of equilibration in vapor-diffusion crystallization

The kinetics of water-vapor equilibration in macromolecular crystallization were investigated for sitting droplets of aqueous polyethylene glycol (PEG) 8000 as a function of concentration. Equilibrations, set up with initial concentrations of PEG in the droplet at half those in the reservoir, were very slow for concentrations of relevance to the macromolecular crystal growth problem. At 301 K, 24 µl droplets at initial concentrations of 2.5, 5.0 and 7.5%(w/v) PEG require 12, 5, and 3 weeks to reach equilibrium, respectively. On the other hand, the addition of modest quantities of sodium chloride to both droplet and reservoir increases the rate of equilibration for aqueous PEG sitting droplets significantly. At 293 K, droplets with initial volumes of 24 µl and PEG concentrations of 5%(w/v) require 12 weeks to reach equilibrium, while droplets of the same volume and initial concentrations of 5%(w/v) PEG and 200 mM NaCI require less than two weeks to reach equilibrium. The slow vapor-diffusion equilibrations of pure PEG solutions, and the subsequent increase in these rates with colligative agents such as salt, are a consequence of the non-ideality of aqueous PEG solutions. These results are of interest both from a practical and a theoretical viewpoint. They underscore the importance of kinetic factors in macromolecular crystal growth, help to explain apparent inconsistencies of outcome in PEG-mediated crystallizations, and yield another methodology for the optimization of crystal growth conditions, namely the control of the kinetics of equilibration using colligative agents.