TY - JOUR
T1 - Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins
AU - Kawate, Toshimitsu
AU - Gouaux, Eric
N1 - Funding Information:
The authors are grateful to H. Furukawa and S.K. Singh for bacterial expression vector construction and critical comments on the manuscript. We would like to thank A. Yamashita, J. Jasti, D. Yernool, and O. Boudker for FSEC examples and figures. We would also like to acknowledge J. Hunt for the GFPuv vector. Diffraction data were collected at the National Synchrotron Light Source and the Advanced Light Source. This work was supported by the National Institutes of Health and the Howard Hughes Medical Institute (HHMI). E.G. is an investigator with the HHMI.
PY - 2006/4
Y1 - 2006/4
N2 - Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.
AB - Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.
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U2 - 10.1016/j.str.2006.01.013
DO - 10.1016/j.str.2006.01.013
M3 - Article
C2 - 16615909
AN - SCOPUS:33646011258
SN - 0969-2126
VL - 14
SP - 673
EP - 681
JO - Structure
JF - Structure
IS - 4
ER -