TY - JOUR
T1 - Quantitative imaging by confocal scanning fluorescence microscopy of protein crystallization via liquid-liquid phase separation
AU - Vivarès, Denis
AU - Kaler, Eric W.
AU - Lenhoff, Abraham M.
PY - 2005/6/1
Y1 - 2005/6/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=23844458001&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=23844458001&partnerID=8YFLogxK
U2 - 10.1107/S090744490402949X
DO - 10.1107/S090744490402949X
M3 - Article
C2 - 15930647
AN - SCOPUS:23844458001
SN - 0907-4449
VL - 61
SP - 819
EP - 825
JO - Acta Crystallographica Section D: Biological Crystallography
JF - Acta Crystallographica Section D: Biological Crystallography
IS - 6
ER -