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N J Ryba

Publications and source records attributed to N J Ryba.

30 records · Page 2Linked to original sources
PubMed1992

The gamma-subunit of the principal G-protein from squid (Loligo forbesi) photoreceptors contains a novel N-terminal sequence.

The squid (Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the gamma-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-gamma has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the alpha- and beta-subunits of this protein. In particular, the N-terminus of the squid visual G-gamma contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-gamma accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.

Amino Acid Sequence↗
PubMed1992

Protein rotational diffusion and lipid/protein interactions in recombinants of bovine rhodopsin with saturated diacylphosphatidylcholines of different chain lengths studied by conventional and saturation-transfer electron spin resonance.

Bovine rhodopsin has been reconstituted in seven different saturated diacylphosphatidylcholine species of odd and even chain lengths from C-12 to C-18 at a lipid/protein ratio (60:1 mol/mol) comparable to that in the native rod outer segment disk membrane. All recombinants were found to be photochemically active, in that optical bleaching produced a temperature- and lipid chain-length-dependent mixture of species absorbing at 480 and 380 nm. Both the rotational diffusion of rhodopsin and lipid-protein interactions in the various recombinants were studied by saturation transfer and conventional electron spin resonance spectroscopy of spin-labeled rhodopsin and of spin-labeled phosphatidylcholine, respectively. In the fluid lipid phase, the rotational diffusion rate of rhodopsin was found to be dependent on the lipid chain length of the different recombinants in a nonmonotonic manner. The diffusion rate in dilauroylphosphatidylcholine was found to be very slow, indicating extensive protein aggregation, whereas that in dipentadecanoylphosphatidylcholine was rapid (effective correlation time ca. 7 microseconds), consistent with the presence of monomeric protein. For recombinants with longer lipid chain lengths, the rotational diffusion rate again decreased, indicating the presence of di- or oligomeric protein. The fraction of lipid motionally restricted at temperatures in the fluid phase was also dependent on the chain length of the phosphatidylcholine used in the reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The identification and purification of the heterotrimeric GTP-binding protein from squid (Loligo forbesi) photoreceptors.

The principal GTP-binding protein (G-protein) from squid (Loligo forbesi) photoreceptor membranes has been identified by amino acid sequencing. The heterotrimeric protein was purified by detergent solubilization and ion-exchange chromatography. The amino acid sequence of the G-protein alpha-subunit (G-alpha) indicates that this subunit is closely related to the recently characterized Gq subgroup, whereas the G-gamma subunit varies widely in sequence from other homologues.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and primary structure of squid (Loligo forbesi) rhodopsin, a phospholipase C-directed G-protein-linked receptor.

The sequence of squid (Loligo forbesi) rhodopsin was determined by protein and cDNA sequencing. The protein has close similarity to octopus rhodopsin, having an N-terminal region (residues 1-340) which resembles other guanine-nucleotide-binding protein (G-protein)-linked receptors and a repetitive proline-rich C-terminus (residues 340-452). Comparison of the sequence of squid rhodopsin with those of other members of the G-protein-linked receptor superfamily reveals features which we predict to have both structural and functional importance.

Amino Acid Sequence↗
PubMed1991

Sequence of the beta-subunit of the phosphatidylinositol-specific phospholipase C-directed GTP-binding protein from squid (Loligo forbesi) photoreceptors.

The beta-subunit (G-beta) of the squid (Loligo forbesi) visual GTP-binding protein (G-protein), thought to be associated with a phosphatidylinositol-specific phospholipase C, has been identified and the sequence of the protein determined from its cDNA. The predicted polypeptide has a very marked sequence similarity with its mammalian counterparts (80-90% identity). Squid G-beta also has somewhat lower similarity to the yeast protein STE4 (approx. 40% identity). The role of G-beta in signal transduction is discussed in the light of its pronounced structural conservation.

Amino Acid Sequence↗
PubMed1990

Phase transition from a gel to a fluid phase of cubic symmetry in dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) bilayers.

Aqueous dispersions (pH 4.0) of a 2:1 (mol/mol) mixture of myristic acid with dimyristoylphosphatidylcholine undergo a sharp transition at 45-47 degrees C from a lamellar gel phase to a fluid phase which is optically isotropic. This fluid phase gives rise to 31P-NMR spectra, and 2H-NMR spectra of the chain-deuterated components, which are also isotropic. X-ray diffraction studies of the fluid phase at 49 degrees C, reveal reflections with spacings in the ratio square root of 2: (square root of 3): square root of 4: square root of 6: square root of 8, accompanied by a strong diffuse scatter. These reflections index on a cubic lattice of primitive space group Pn3 or Pn3m, or possibly the body-centered group Im3m, with a lattice constant of 21.2 nm. The dimensions of the phase are consistent with a structure composed of two systems of tetrahedrally (octahedrally) oriented inverted lipid cylinders, found for other cubic lipid phases with Pn3m (Im3m) symmetry. At higher temperatures the cubic phase gradually converts, with increasing temperature, to a coexisting inverted hexagonal phase.

Dimyristoylphosphatidylcholine↗
PubMed1987

Molecular exchange at the lipid-rhodopsin interface: spin-label electron spin resonance studies of rhodopsin-dimyristoylphosphatidylcholine recombinants.

The photoreceptor protein rhodopsin has been reconstituted with a single phospholipid species, dimyristoylphosphatidylcholine, at a range of different lipid/protein ratios, and the exchange rate at the lipid-protein interface has been determined from the electron spin resonance spectra of spin-labeled phosphatidylcholine. For recombinants with lipid/protein ratios in the range 41:1 to 102:1 (mol/mol), the electron spin resonance spectra of 1-acyl-2-[14-(4,4-dimethyloxazolidine-N-oxyl)stearoyl]-sn-glycero-3- phosphocholine consist of a fluid component similar to that found in pure lipid bilayers and a motionally restricted component corresponding to lipids whose motion is reduced by interaction with the intramembranous surface of rhodopsin. The relative proportion of the motionally restricted component increases with increasing protein content in the complex. Spectral subtraction with fluid and motionally restricted components (from fluid- and gel-phase lipid, respectively), which best fit the apparent components in the complex, reveals that 22 +/- 2 lipids per 39,000-dalton protein are motionally restricted, independent of lipid/protein ratio and of temperature. Simulation of the two-component spectra with the exchanged-coupled Bloch equations gives values for both the fraction of motionally restricted component and the exchange rate between the two components. Using fixed motionally restricted and fluid component line shapes at a given temperature, it is possible to obtain a consistent description of the lipid/protein ratio dependence of the spectra at each temperature. The number of motionally restricted lipids obtained by simulation, allowing for exchange, is 23 +/- 3 per 39,000-dalton protein, again independent of temperature and of lipid/protein ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Protein-lipid interactions at membrane surfaces: a deuterium and phosphorus nuclear magnetic resonance study of the interaction between bovine rhodopsin and the bilayer head groups of dimyristoylphosphatidylcholine.

Rhodopsin, isolated from bovine retinal rod outer segment disk membranes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine which was deuterated in the terminal methyl groups of the choline polar head group. By use of a mixed detergent system of cholate and octyl glucoside to solubilize the phospholipid and rhodopsin, 15 membrane complexes of predetermined phospholipid to rhodopsin mole ratios of between 350:1 and 65:1 have been produced by exhaustive dialysis and studied by a variety of techniques. Electron micrographs of replicas from freeze-fractured membrane complexes showed that the majority of the lipid, for all rhodopsin:phospholipid ratios, was contained in large bilayer vesicles with diameters in excess of 400 nm. Complexes produced with rhodopsin from frozen retina produced an absorption maximum at 478 nm after photobleaching whereas rhodopsin from fresh retina could be bleached more completely to an absorption maximum at 380 nm. Deuterium nuclear magnetic resonance (NMR) spectra from the lipid head groups of bilayers above the gel to liquid-crystalline phase transition temperature were shown to be sensitive in a systematic way to the presence of rhodopsin which could be bleached to 380 nm. The measured quadrupole splittings, taken as the separation of the turning points of the recorded NMR spectra, decreased from a value of 1.28 kHz for protein-free bilayers to approximately 0.40 kHz for bilayers containing 65 molecules of phospholipid for each rhodopsin at 32 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Evidence from deuterium nuclear magnetic resonance for the temperature-dependent reversible self-association of erythrocyte band 3 in dimyristoylphosphatidylcholine bilayers.

Band 3, isolated from human erythrocytes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) deuterated in the terminal methyl groups of the choline head group. By use of Triton X-100 for selective extraction and purification of band 3 and then cholate for subsequent solubilization with the lipid, a number of reconstituted complexes were produced by exhaustive detergent dialysis with protein:lipid weight ratios of between 0.32:1 and 1.25:1. Electron micrographs of negatively stained complexes showed that this method produced large vesicles of greater than 300-nm diameter. Deuterium nuclear magnetic resonance (NMR) spectra from the choline methyl deuterons in bilayer lipid above the liquid-crystal-gel phase transition temperature were shown to change systematically with increasing concentrations of band 3 in the bilayers. The measured quadrupole splittings, taken as the separation of the turning points in the recorded spectra, decreased from a value of 1.28 kHz for pure lipid to 0.98 kHz for bilayers with a protein:lipid ratio of 1.25:1 at 26 degrees C. At 35 degrees C, a more pronounced decrease in the quadrupole splittings was measured. The data from the complexes with protein:lipid ratios up to 0.7:1 (w/w) obey the mathematical treatment for a rapid two-site exchange between lipids at the protein-lipid interface and the bulk lipid phase. The temperature dependence of the measured quadrupole splitting with respect to the protein:lipid ratio indicates that the amount of lipid at the protein-lipid interface increases with increasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Anion Exchange Protein 1, Erythrocyte↗
PubMed1985

Effect of calmodulin on the structural state of photoreceptor membranes and rhodopsin-containing phospholipid vesicles.

The effect of calmodulin on the order of lipids in rhodopsin-free and rhodopsin-containing membranes has been studied using spin-label electron spin resonance methods. Calmodulin, up to 10(-6)M, did not change the measured order of lipids in bilayer membranes containing only rhodopsin. However, for bovine rod outer segment disc membranes, which contain rhodopsin and other proteins, calmodulin induced a significant concentration and temperature dependent increase in the order of the membrane lipids. This suggests that the site of calmodulin binding is remote from rhodopsin itself, and the nature of the binding appears to be a membrane surface phenomenon.

Animals↗