Refolding of bacteriorhodopsin

Abstract

Staphylococcus aureus protease V8 cleaves bacteriorhodopsin to two main fragments, V-I and V-2. Proteolytic digestion of the purple membrane integrated protein is carried out in the presence of limited amounts of sodium dodecyl sulfate (0.5 g detergent/g bacteriorhodopsin). The fragment V-1 includes the arylisothiocyanate binding site (Lys41). The V-2 fragment comprises the two C-terminal transmembrane segments of bacteriorhodopsin. Improved renaturation of bacteriorhodopsin and the ternary complex, reformed from its V8 proteolytic fragments, is attained by peptide extraction in chloroform/methanol/O.l M ammonium acetate and subsequent incorporation into phospholipid/detergent micelles. In the presence of retinal, V8 fragments reform chromophoric ternary complexes. Light-adapted reconstituted chromophores absorb incident light at 560 nm. Protein secondary structures are partially conserved in the course of solvent extraction and are restored in the reconstituted system. Vesicles prepared from the reconstituted complexes show light-dependent proton translocation activity.Light-induced proton translocation across the purple membrane of Hafobacierium hnfobium is catalyzed by the retinylidene protein bacteriorhodopsin (reviewed in [l]). Understanding the mechanism by which protons are transported through the mediating bacteriorhodopsin molecule requires knowledge of the spacial arrangement of its seven transmembrane (helical) segments [2]. Cognizance of the chemical properties of the components participating in the transport process is essential. Crystallization and diffraction studies [3 -51 in conjunction with selective modulation of minimal regions within the protein, e.g. by site-directed mutagenesis [6] or semisynthetic procedures, may help to identify functionally relevant structures. All approaches mentioned depend on the correct folding of the manipulated polypeptide chain. The tertiary structure of bacteriorhodopsin is remarkably stable [7]. Its refolding capacity is exceptional. Following complete denaturation in protic solvents, uncleaved bacteriorhodopsin readily refolds and, upon addition of retinal and a phospholipid/detergent mixture followed by dialysis, it forms fully active vesicles [8,9]. Native bacteriorhodopsin-like structures can be regenerated from chymotryptic fragments [8, 10, 111. The fragments associate to crystalline purple membrane structures [12]. Partial refolding of native bacteriorhodopsin structures has also been attained with fragments produced by NaBH4 reduction of bacteriorhodopsin. This treatment cleaves the Glyl55 -Phel56 peptide bond in the primaryCorrespondence to H. Sigrist, Institute of Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland This paper is dedicated to Prof. G. Semenza on the occasion of his 60th birthday.Abbreviations. PITC, phenylisothiocyanate, PTC-, phenylthiocarbamoyl-; DABITC, 4-N,N'-dimethylamino-azobenzene-4-isothiocyanate; NBD-CI, 7-chloro-4-nitrobenz-2-oxa-I ,3-diazole; MyrzGroPCho, dimyristoylglycerophosphocholine; solvent ...

Invited review GPCR structural characterization: Using fragments as building blocks to determine a complete structure

“…The Dumont group coexpressed fragments of Ste2p in yeast and found that these fragments reconstituted in the membrane to form a functional receptor . In vivo reconstitution has also been successful for the muscarinic M2 and M3 receptors and the dopamine D 2s receptors and in vitro reconstitution in detergent micelles was reported for the GPCR analogue bacteriorhodopsin . Based on these results many groups have been studying fragments of membrane protein receptors to better understand their structure‐function relationships.…”

“…The protein was completely denatured and then refolded into a functional protein. Initial fragment analysis was performed by digesting bacteriorhodopsin with either V8 protease or chymotrypsin, purifying the fragments and then reconstituting with complementary fragments and/or chemically synthesized fragments in micelles or H. halobium lipids . A large body of work that covered individual and multi‐TM fragments of bacterioopsin came from the Shemyakin Institute of Bioorganic Chemistry in Russia .…”