Purification, cloning and expression of a signal transducer for sensory rhodopsin I in Halobacterium salinarium

Date

1993

Authors

Yao, Virginia Jean

Journal Title

Journal ISSN

Volume Title

Publisher

ProQuest Dissertations & Theses

YU Faculty Profile

Abstract

Sensory rhodopsin I (SR-I) is a retinylidene integral membrane protein that functions as a photoreceptor in the archaeon, Halobacterium salinarium. SR-I modulates the frequency of swimming direction reorientation allowing cells to swim toward light beneficial for energy production and away from damaging near-UV radiation. Biochemical and mutant analyses identified a reversibly methylated membrane protein of M{dollar}\sb{lcub}\rm r{rcub}{dollar} 97 kDa necessary for SR-I signalling. These results suggested a similar function of this halophilic protein to the eubacterial methyl-accepting chemotaxis signal transducer membrane proteins.;To study the role of the 97 kDa M{dollar}\sb{lcub}\rm r{rcub}{dollar} protein (named HtrI for the halobacterial transducer for SR-I) in the SR-I signalling pathway, N-terminal and two tryptic peptide sequences were obtained from SDS-PAGE purified HtrI. Three fully degenerate deoxyoligonucleotides were designed to sequences within one tryptic peptide to clone htrI from a genomic library. Double-stranded sequencing of htrI revealed it resides directly upstream and adjacent to the SR-I gene, sopI. The translated molecular weight of HtrI is 57 kDa with an isoelectric point of 3.9. Kyte/Doolittle analysis of the deduced protein sequence predicts two putative transmembrane regions near the N-terminus, and the remainder of the protein (536 amino acid residues total) is hydrophilic. HtrI exhibits 64% identity and 87% sequence similarity to the highly conserved region within the signalling domain of the eubacterial chemotaxis signal transducer, Tsr, and flanking methylation domains retain essential methylation sites as well. Hence, the predicted structure, gene organization, and sequence similarity to the methylation and signalling domains of the eubacterial proteins support the role of HtrI as the SR-I signal transducer.;Transformation of an H. salinarium phototaxis null mutant strain with htrI/sopI utilized an autonomously replicating selectable expression vector. Characterization of the htrI/sopI transformant swimming behavior by motion analysis to SR-I specific photostimuli and flash photolysis indicate complete restoration of SR-I mediated phototaxis.;Expression of htrI in E. coli revealed that {dollar}>{dollar}90% is recoverable in the soluble fraction. Affinity chromatography purified HtrI will be used to generate polyclonal antibodies and to initiate in vitro reconstitution studies.

Description

Keywords

Molecular biology., Biochemistry., Microbiology.

Citation

Source: Dissertation Abstracts International, Volume: 54-04, Section: B, page: 1814.;Advisors: John L. Spudich.