Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants.

Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.

[1]  G. Semenza,et al.  Membrane protein topology: amino acid residues in a putative transmembrane .alpha.-helix of bacteriorhodopsin labeled with the hydrophobic carbene-generating reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine , 1985 .

[2]  H. Khorana,et al.  Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[3]  W. Plachy,et al.  The diffusion-solubility of oxygen in lipid bilayers. , 1980, Biochimica et biophysica acta.

[4]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[5]  K. Wüthrich Protein structure determination in solution by nuclear magnetic resonance spectroscopy. , 1989, Science.

[6]  Y. Ovchinnikov Probing the folding of membrane proteins , 1987 .

[7]  H. Khorana,et al.  Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212. , 1988, Biochemistry.

[8]  H. Khorana Bacteriorhodopsin, a membrane protein that uses light to translocate protons. , 1988, The Journal of biological chemistry.

[9]  H. Guy Amino acid side-chain partition energies and distribution of residues in soluble proteins. , 1985, Biophysical journal.

[10]  D. Eisenberg Three-dimensional structure of membrane and surface proteins. , 1984, Annual review of biochemistry.

[11]  H. Khorana,et al.  Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[12]  W. Lim,et al.  Alternative packing arrangements in the hydrophobic core of λrepresser , 1989, Nature.

[13]  H. Khorana,et al.  Immunological probes for bacteriorhodopsin. Identification of three distinct antigenic sites on the cytoplasmic surface. , 1982, The Journal of biological chemistry.

[14]  H. Khorana,et al.  Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F. , 1987, The Journal of biological chemistry.

[15]  S. Englander,et al.  Quenching of room temperature protein phosphorescence by added small molecules. , 1988, Biochemistry.

[16]  H. Khorana,et al.  Aspartic acid substitutions affect proton translocation by bacteriorhodopsin. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[17]  D. Engelman,et al.  Path of the polypeptide in bacteriorhodopsin. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[18]  H. Khorana,et al.  Structure-function studies on bacteriorhodopsin. IV. Purification and renaturation of bacterio-opsin polypeptide expressed in Escherichia coli. , 1987, The Journal of biological chemistry.

[19]  J. S. Hyde,et al.  Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation recovery and continuous wave power saturation electron paramagnetic resonance. , 1989, Biophysical journal.

[20]  G. Büldt,et al.  Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing. , 1989, Biochimica et biophysica acta.

[21]  D Eisenberg,et al.  Hydrophobic organization of membrane proteins. , 1989, Science.

[22]  J. S. Hyde,et al.  The diffusion-concentration product of oxygen in lipid bilayers using the spin-label T1 method. , 1981, Biochimica et biophysica acta.

[23]  H. Khorana,et al.  Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines. , 1989, Biochemistry.

[24]  Khorana Hg,et al.  Structural studies on transmembrane proteins. 1. Model study using bacteriorhodopsin mutants containing single cysteine residues. , 1989 .

[25]  C. Levinthal,et al.  Site‐directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation , 1989, Proteins.

[26]  H. Khorana,et al.  Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin. , 1989, The Journal of biological chemistry.

[27]  D. Engelman,et al.  Tertiary structure of bacteriorhodopsin. Positions and orientations of helices A and B in the structural map determined by neutron diffraction. , 1989, Journal of molecular biology.

[28]  R. Henderson,et al.  Three-dimensional model of purple membrane obtained by electron microscopy , 1975, Nature.

[29]  J. Rigaud,et al.  Monomer-oligomer equilibrium of bacteriorhodopsin in reconstituted proteoliposomes. A freeze-fracture electron microscope study. , 1987, The Journal of biological chemistry.

[30]  J. Deisenhofer,et al.  The Photosynthetic Reaction Center from the Purple Bacterium Rhodopseudomonas viridis , 1989, Science.

[31]  W. Stoeckenius,et al.  Bacteriorhodopsin and related pigments of halobacteria. , 1982, Annual review of biochemistry.