Quantitative imaging by confocal scanning fluorescence microscopy of protein crystallization via liquid-liquid phase separation

Denis Vivarès, Eric W. Kaler, Abraham M. Lenhoff

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)819-825
Number of pages7
JournalActa Crystallographica Section D: Biological Crystallography
Volume61
Issue number6
DOIs
StatePublished - Jun 1 2005

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