The 3.7 Å projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer

GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine‐tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of ∼80 Å side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two‐dimensional crystals, which diffracted electrons to 3.6 Å resolution. Cryoelectron microscopy provided a 3.7 Å projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.

[1]  W. O. Saxton,et al.  The major cell envelope protein of Micrococcus radiodurans (R1). Structural and chemical characterization. , 1982, European journal of biochemistry.

[2]  R. Henderson,et al.  Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. , 1990, Journal of molecular biology.

[3]  P. Agre,et al.  Erythrocyte Mr 28,000 transmembrane protein exists as a multisubunit oligomer similar to channel proteins. , 1991, The Journal of biological chemistry.

[4]  J. Lepault,et al.  Structure of purple membrane from halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 Å resolution , 1986 .

[5]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[6]  P. Postma,et al.  Trehalose transport and metabolism in Escherichia coli , 1990, Journal of bacteriology.

[7]  D. Thomas,et al.  Prediction of functional residues in water channels and related proteins , 1998, Protein science : a publication of the Protein Society.

[8]  P. Agre,et al.  Molecular structure of the water channel through aquaporin CHIP. The hourglass model. , 1994, The Journal of biological chemistry.

[9]  A. Cheng,et al.  Three-dimensional organization of a human water channel , 1997, Nature.

[10]  M. Yeager,et al.  The CHIP28 water channel visualized in ice by electron crystallography , 1995, Nature Structural Biology.

[11]  D Kozono,et al.  Functional reconstitution and characterization of AqpZ, the E. coli water channel protein. , 1999, Journal of molecular biology.

[12]  Huilin Li,et al.  Molecular design of aquaporin-1 water channel as revealed by electron crystallography , 1997, Nature Structural Biology.

[13]  Y. Fujiyoshi,et al.  The structure of aquaporin-1 at 4.5-A resolution reveals short alpha-helices in the center of the monomer. , 1999, Journal of structural biology.

[14]  M. Saier,et al.  The MIP family of integral membrane channel proteins: sequence comparisons, evolutionary relationships, reconstructed pathway of evolution, and proposed functional differentiation of the two repeated halves of the proteins. , 1993, Critical reviews in biochemistry and molecular biology.

[15]  J B Heymann,et al.  Structural clues in the sequences of the aquaporins. , 2000, Journal of molecular biology.

[16]  Andreas Engel,et al.  Aquaporins: Phylogeny, Structure, and Physiology of Water Channels. , 1999, News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society.

[17]  M. Saier,et al.  Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. , 1994, The Journal of biological chemistry.

[18]  J Frank,et al.  Three-dimensional reconstruction of single particles embedded in ice. , 1992, Ultramicroscopy.

[19]  M. Yeager,et al.  Projection structure of a plant vacuole membrane aquaporin by electron cryo-crystallography. , 1999, Journal of molecular biology.

[20]  A. Engel,et al.  Factors influencing the precision of quantitative scanning transmission electron microscopy , 1992 .

[21]  R Henderson,et al.  Analysis of electron microscope images and electron diffraction patterns of thin crystals of phi 29 connectors in ice. , 1994, Journal of molecular biology.

[22]  T. Walz,et al.  The three‐dimensional structure of human erythrocyte aquaporin CHIP. , 1994, The EMBO journal.

[23]  Andreas Engel,et al.  The three-dimensional structure of aquaporin-1 , 1997, Nature.

[24]  S. Deschamps,et al.  Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis. , 1999, Journal of structural biology.

[25]  W. Boos,et al.  Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator , 1993, Journal of bacteriology.

[26]  Müller,et al.  Mass Measurement in the Scanning Transmission Electron Microscope: A Powerful Tool for Studying Membrane Proteins. , 1998, Journal of structural biology.

[27]  M. Borgnia,et al.  Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography. , 1999, Journal of molecular biology.

[28]  P. Tittmann,et al.  Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution. , 1998, Journal of molecular biology.

[29]  S. Deschamps,et al.  Switch from an Aquaporin to a Glycerol Channel by Two Amino Acids Substitution* , 1999, The Journal of Biological Chemistry.

[30]  J. H. Park,et al.  Phylogenetic Characterization of the MIP Family of Transmembrane Channel Proteins , 1996, The Journal of Membrane Biology.

[31]  J. Knights.  Structural and chemical characterization , 1984 .

[32]  A. Engel,et al.  Projection map of aquaporin-1 determined by electron crystallography , 1995, Nature Structural Biology.

[33]  U Aebi,et al.  2D crystallization: from art to science. , 1992, Ultramicroscopy.

[34]  S. Deschamps,et al.  Oligomerization State of Water Channels and Glycerol Facilitators , 1998, The Journal of Biological Chemistry.

[35]  Y. Fujiyoshi,et al.  Trehalose embedding technique for high-resolution electron crystallography: application to structural study on bacteriorhodopsin. , 1999, Journal of electron microscopy.