Quantitative imaging by confocal scanning fluorescence microscopy of protein crystallization via liquid-liquid phase separation.
Metastable states such as liquid-liquid phase separation, aggregation and gelation can affect protein crystallization but their positive or negative effects are only partially understood. In this work, mixtures of PEG (MW 10 kDa) and a large model protein, glucose isomerase (MW 173 kDa), have been studied to characterize the effect of a metastable liquid-liquid phase separation on protein crystallization. Fluorescence labeling allowed confocal fluorescence microscopy observations and quantification of the partitioning of the protein and PEG between the liquid phases and showed two steps in the crystallization process. Two crystallization mechanisms within the liquid domain were revealed, yielding two different polymorphs. With one polymorph, few crystals nucleated and grew droplet-by-droplet in the dispersed concentrated liquid phase, while for the other homogeneous crystal nucleation and growth occurred independently and simultaneously in numerous droplets of the concentrated phase. The results demonstrate the substantial possible complexity of crystallization behavior, as well as its sensitivity to the location of the conditions on the phase diagram and to the physicochemical properties of the system.
Vivarès
Denis
D
Center for Molecular and Engineering Thermodynamics, Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.
Kaler
Eric W
EW
Lenhoff
Abraham M
AM
D019747:Aldose-Ketose Isomerases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods; D013302:Streptomyces / Q000201:enzymology*
D001426:Bacterial Proteins; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase