Yu.A. Ovchinnikov

The structural basis of the functioning of bacteriorhodopsin: an overview.

Mass transport across biological membranes is a pressing problem of modern physicochemical biology, determining to a considerable extent our understanding of the regulatory and bioenergetic processes in the living cell. Latterly, as well as continued interest in the general aspects and theories of transport and in the study of model systems, one may clearly discern a tendency in this field towards detailed investigation of the proteins directly associated with translocation of ions and molecules, independently of whether in the membrane such proteins serve as carriers, channels or receptors. Such a membrane protein, one of the most interesting and extensively explored, undoubtedly is bacteriorhodopsin. Bacteriorhodopsin, the only protein of the purple membrane of halophilic bacteria which thrive under extremal conditions and effectively utilize light energy in the performance of their vital activity [ 11, has been found to function as a light-driven proton pump, establishing a considerable pH gradient in the membrane, utilized in the synthesis of ATP [2]. Despite available source and relatively small size, bacteriorhodopsin has long foiled attempts to elucidate its structure by numerous protein chemists and physicists, and only very recently has a way been found to decode its amino acid sequence and elucidate the chemical nature of its functionally important groups [3,17]. These recent studies have also given the first indications of how the molecule is packed in the membrane. The present paper is a summing up of our studies, begun in 1976, that have resulted in the complete

Two adjacent cysteine residues in the C-terminal cytoplasmic fragment of bovine rhodopsin are palmitylated.

Covalent coupling of bovine rhodopsin to CPG‐thiol glass was used for separation of CNBr peptides. It is shown that cysteine residues 322 and 323 in the C‐terminal cytoplasmic fragment of rhodopsin are modified with palmitic acid.

Rhodopsin and bacteriorhodopsin: structure—function relationships

Photoreception and light transformation by living systems undoubtedly belong to the most surprising natural phenomena. Along with low-M, chromophoric compounds, specific proteins play an important part in absorption and transduction of light energy. Among photosensitive proteins, rhodopsin occupies a key position. This chromoprotein, with 1 l-c&retinal as a photoreceptor antenna, is the basis of the vision process in animals. For a long time all attempts to isolate rhodopsin from the photosensitive disc membranes in an individual and active state were not a success. The preparations obtained with various detergents were unsuitable for detailed structural analysis. Though dozens of laboratories actively participated in investigations of rhodopsin function and its structure peculiarities [l-6], the progress was rather slow. gradient of H+ turned out to be a universal energetic source for the cell. Interestingly, full protonpumping activity has been restored after complete delipidation and following reconstitution of bacteriorhodopsin into liposomes [ 16-l 81.

Conformational studies of peptide systems : The rotational states of the NHCH fragment of alanine dipeptides by nuclear magnetic resonance

Abstract The IR and NMR-H 1 spectra of alanine dipeptides and their N-Me derivatives have been investigated. Measurement of the integral intensities of the NH stretching vibrations in dilute solutions of CCl 4 and of a (1:9) CHCl 3 + CCl 4 mixture showed that in these solvent approximately 70% of the alanine dipeptide molecules are in an intramolecular hydrogen-bonded folded form. It was found from analysis of the vicinal proton spin-spin coupling constant of the CONHCH fragment that the stable conformer with respect to this bond is that with cis -arrangement of the NH and CH hyrogens. The conformation of the 7-membered hydrogen-bonded ring of the dipeptides has been elucidated. An empirically found stereochemical dependence of the constant 3 J NHCH upon the dihedral angle θ of the fragment has served as basis for discussing the possible conformations of the extended form of the dipeptides in polar (including aqueous) solvents.

1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices

The structure of[Val1]gramicidin A incorporated into sodium dodecyl‐d 25 sulphate micelles has been studied by two‐dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin‐spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N‐terminal to N‐terminal (head‐to‐head) dimer formed by two right‐handed, single‐stranded β6.3 helices with 6.3 residues per turn, differing from Urrys structure by handedness of the helices.

Refinement of the angular dependence of the peptide vicinal NHCαH coupling constant

Abstract The refined dependence of the peptide NHCαH vicinal coupling constant on the dihedral angle θ have been derived on the basis of the accumulated experimental data. The mean permissible values (in Hz) are approximated by 3 J NHCH = 9·4 cos 2 θ - 1·1 cos θ + 0·4 An analogous relationship for the sum of two vicinal NHCαH 2 coupling constants in the glycyl residue have been calculated from the above dependence. Measurements on N-methylacetamide in various solvents and in the presence of an alkali salt showed the vicinal constant NHCH to vary by not more than ± 3%. Some of the other proposed 3 J NHCH (θ) dependencies give too low values for the cis -oriented NH and CαH bonds. This may be due to the fact that in these correlations the data for compounds with cis -amide bonds have been used for 0° ⩽ θ ⩽ 90° region of the dependence.

Pig kidney Na+,K+-ATPase: Primary structure and spatial organization

(Na+ + K+)‐ATPase α‐Subunit β‐Subunit cDNA nucleotide sequence Primary structure Glycopeptide Transmembrane arrangement

The complete amino acid sequence of cytoplasmic aspartate aminotransferase from pig heart

Aspartate aminotransferase (L-aspartate: 2-0x0glutarate aminotransferase, EC 2.6.1.1) is one of the principal pyridoxal-P-containing enzymes that catalyse the transamination reactions [3] representing key steps at the intersection between the metabolic pathways of amino acids and dicarboxylic acids. Although the catalytic mechanism of aspartate aminotransferase has been investigated at the level of substrate-coenzyme models [4], its elucidation in detail requires knowledge of the enzyme’s structure, considering, in particular, that the very high rates of the enzymic process are determined by the structural peculiarities of the specific protein(apoenzyme) of the aspartate aminotransferase. Accordingly, we embarked on the task of elucidating the amino acid sequence of this enzyme. In the present paper the concluding stage of the work is reported*. The object chosen for study was the aspartate aminotransferase of the cytosol of pig heart; the enzyme, which is different from the mitochondrial isozyme [5,6] was prepared by a previously reported procedure [7]. The enzyme is a complex dimeric protein of high molecular weight; each of the associated subunits

Octopus rhodopsin Amino acid sequence deduced from cDNA

The primary structure of rhodopsin from the octopus Paroctopus defleini has been determined by parallel analysis of the protein and corresponding cDNA. The amino acid sequence is most similar to the recently cloned Drosophila opsins. Similarities to bovine and human opsins are also evident. The transmembrane topology of octopus rhodopsin is discussed.

Cyclic GMP phosphodiesterase from bovine retina Amino acid sequence of the α-subunit and nucleotide sequence of the corresponding cDNA

The primary structure of the γ‐subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme γ‐subunit contains 87 amino acid residues, its N‐terminal amino group being acetylated.