N.G. Abdulaev

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.

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.

Photosynthetic reaction centre of Chloroflexus aurantiacus. I. Primary structure of L-subunit.

The L‐subunit primary structure of the reaction centre from Chloroflexus aurantiacus composed of 310 amino acid residues has been determined by parallel analysis of the protein and corresponding DNA. Significant homology between this protein and L‐subunits from reaction centres of purple bacteria is observed. This implies close similarity in the tertiary structure of these proteins.

PRODUCTS OF LIMITED PROTEOLYSIS OF BACTERIORHODOPSIN GENERATE A MEMBRANE POTENTIAL

In recent years, one of the most interesting events in biochemistry was the discovery of a new type of photosynthesis mediated by bacteriorhodopsin, a light-dependent proton pump in the membrane of halophilic bacteria. The goal of the present work was to elucidate whether the water-exposed sites of the polypeptide chain of bacteriorhodopsin are involved in the generation of a transmembrane electric potential difference. We have studied the products of partial proteolysis of bacteriorhodopsin by papain and thermolysin. The experiments revealed that the products of hydrolysis of bacteriorhodopsin that retain the AsTO,, max are still competent in generating a transmembrane electric potential difference if illuminated with continuous light or with a flash inducing a single bacteriorhodopsin turnover. The kinetic parameters of bacteriorhodopsin as a photoelectric generator do not deteriorate in spite of removal of 17 amino acids from the C-end, 3 amino acids from the N-end and 5 amino acids from the middle part of the polypeptide chain.

P26 — calcium binding protein from bovine retinal photoreceptor cells

The primary structure of bovine retinal calcium binding protein P26 has been determined by parallel analysis of protein and corresponding cDNA, This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. Preliminary data are presented on expression of P26 as a fusion protein in E. coli.

Cytoplasmic aspartate aminotransferase from pig heart muscle: Partial sequence

In an earlier paper [ 1 ] we reported sequence studies on a number of peptides from the tryptic digest of cytoplasmic aspartate aminotransferase (L-aspartate:2_ketoglutarate aminotransferase, EC 2.6.1.1) from pig heart muscle. Amino acid sequences of a further series of tryptic peptides have been determined, some partial sequences have been completed and data are given on isolation and sequence determination of the products of chymotryptic digestion and cyanogen bromide cleavage of CM-AAT.

The water‐exposed C‐terminal sequence of bacteriorhodopsin does not affect H+ pumping

The fast kinetics of photocycle and H+‐pumping activity of papain‐treated bacteriorhodopsin deprived of 17 C‐terminal amino acid residues has been investigated. As demonstrated by a single‐turnover study, the formation and decomposition of the M412 intermediate as well as the generation of the photoelectric potential are similar in the native and in the papain‐treated protein. On the other hand, acidification of the medium caused by the deprotonation of bacteriorhodopsin due to M412 formation is much smaller in the C‐tail‐deprived protein. Short‐term sonication or addition of a small amount of detergent completely abolishes this effect. As a result, papain‐treated bacteriorhodopsin exhibits the same acidification as the native one. It is concluded that a decrease in the light‐induced pH response of the C‐tail‐deprived bacteriorhodopsin is caused by the aggregation of purple sheets rather than by a special role of the C‐terminal sequence in H+ pumping.

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus.

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone‐10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash‐induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+Q− A) formation. Since the C aurantiacus RC lacks H‐subunit, this subunit was not an obligatory component for electrogenic QB protonation.