Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins.
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.
Kawate
Toshimitsu
T
Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168(th) Street, New York, New York 10032, USA.
Gouaux
Eric
E
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002460:Cell Line; D002850:Chromatography, Gel / Q000379:methods*; D018360:Crystallography, X-Ray; D003902:Detergents / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D018377:Neurotransmitter Agents / Q000378:metabolism; D016011:Normal Distribution; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D018048:Receptors, Purinergic P2 / Q000737:chemistry; D058469:Receptors, Purinergic P2X; D013997:Time Factors
D001426:Bacterial Proteins; D003902:Detergents; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D018377:Neurotransmitter Agents; D011506:Proteins; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D049452:Green Fluorescent Proteins