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.

[1]  E. Campbell,et al.  Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K+ Channel , 2005, Science.

[2]  S. Royle,et al.  P2X Receptor Trafficking in Neurons Is Subunit Specific , 2002, The Journal of Neuroscience.

[3]  R. Garavito,et al.  Detergents as Tools in Membrane Biochemistry* , 2001, The Journal of Biological Chemistry.

[4]  A. Sands,et al.  Knockouts model the 100 best-selling drugs—will they model the next 100? , 2003, Nature Reviews Drug Discovery.

[5]  N. Nomura,et al.  Structural Genomics of Membrane Proteins , 2003 .

[6]  Steven L. Cohen,et al.  Probing the solution structure of the DNA‐binding protein Max by a combination of proteolysis and mass spectrometry , 1995, Protein science : a publication of the Protein Society.

[7]  R D Ricker,et al.  Fast, reproducible size-exclusion chromatography of biological macromolecules. , 1996, Journal of chromatography. A.

[8]  G. Friso,et al.  A scalable, GFP‐based pipeline for membrane protein overexpression screening and purification , 2005, Protein science : a publication of the Protein Society.

[9]  P. Schoenmakers,et al.  Band broadening in size-exclusion chromatography of polydisperse samples. , 2004, Journal of chromatography. A.

[10]  C. Tate,et al.  Overexpression of mammalian integral membrane proteins for structural studies , 2001, FEBS letters.

[11]  Brian Seed,et al.  Codon usage limitation in the expression of HIV-1 envelope glycoprotein , 1996, Current Biology.

[12]  L. Schild,et al.  Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure. , 2002, Physiological reviews.

[13]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

[14]  J. Kendrew.  Preliminary X-ray data for horse and whale myoglobins , 1948 .

[15]  R. North.  Molecular physiology of P2X receptors. , 2002, Physiological reviews.

[16]  W. Stemmer,et al.  Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling , 1996, Nature Biotechnology.

[17]  H. Michel,et al.  Crystallization of membrane proteins. , 1983, Current opinion in structural biology.

[18]  J. Kendrew.  The crystal structure of horse met-myoglobin - I. General features: the arrangement of the polypeptide chains , 1950, Proceedings of the Royal Society of London. Series A. Mathematical and Physical Sciences.

[19]  L. Schild,et al.  The heterotetrameric architecture of the epithelial sodium channel (ENaC) , 1998, The EMBO journal.

[20]  Peter Kuhn,et al.  The genesis of high-throughput structure-based drug discovery using protein crystallography. , 2002, Current opinion in chemical biology.

[21]  J. Møller,et al.  Interaction of membrane proteins and lipids with solubilizing detergents. , 2000, Biochimica et biophysica acta.

[22]  Harren Jhoti,et al.  High-throughput crystallography for lead discovery in drug design , 2002, Nature Reviews Drug Discovery.

[23]  G. von Heijne,et al.  Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli , 2001, FEBS letters.

[24]  B. Khakh.  Molecular physiology of p2x receptors and atp signalling at synapses , 2001, Nature Reviews Neuroscience.

[25]  O. Shimomura,et al.  Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. , 1962, Journal of cellular and comparative physiology.

[26]  B. Boyes,et al.  Size Exclusion Chromatography , 1988 .

[27]  M. Hamon,et al.  Neurotransmitter transporters in the central nervous system. , 1999, Pharmacological reviews.

[28]  R. Tsien,et al.  Creating new fluorescent probes for cell biology , 2002, Nature Reviews Molecular Cell Biology.

[29]  Marie Tichá,et al.  Some potentialities and drawbacks of contemporary size-exclusion chromatography. , 2003, Journal of biochemical and biophysical methods.

[30]  Paul Curnow,et al.  Membrane proteins, lipids and detergents: not just a soap opera. , 2004, Biochimica et biophysica acta.

[31]  Wayne A Hendrickson,et al.  Optimization of protein production in mammalian cells with a coexpressed fluorescent marker. , 2004, Structure.

[32]  W A Hendrickson,et al.  Synchrotron crystallography. , 2000, Trends in biochemical sciences.

[33]  J. Kendrew,et al.  The crystal structure of myoglobin II. Finback whale myoglobin , 1956, Proceedings of the Royal Society of London. Series A. Mathematical and Physical Sciences.

[34]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[35]  E. Gouaux,et al.  Structure of a glutamate transporter homologue from Pyrococcus horikoshii , 2004, Nature.

[36]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[37]  J. Kendrew,et al.  The crystal structure of myoglobin III. Sperm-whale myoglobin , 1957, Proceedings of the Royal Society of London. Series A. Mathematical and Physical Sciences.

[38]  Eric Gouaux,et al.  Crystal structure of LEUTAA, a bacterial homolog of Na+/Cl--dependent neurotransmitter transporters , 2005 .