Tertiary Interactions between Transmembrane Segments 3 and 5 near the Cytoplasmic Side of Rhodopsin

Abstract

Previous studies [Yu, H., Kono, M., and Oprian, D. D. (1999) Biochemistry 38, xxxx-xxxx] using split receptors and disulfide cross-linking have shown that native cysteines 140 and 222 on the cytoplasmic side of transmembrane segments (TM) 3 and 5 of rhodopsin, respectively, can cross-link to each other upon treatment with the oxidant Cu(phen)3(2+). In this paper we show that although the 140-222 cross-link does not affect the spectral properties of rhodopsin, it completely and reversibly inactivates the ability of the receptor to activate transducin. Following on this lead we further investigate the cytoplasmic region of TM3 and TM5 and identify three additional pairs of residues that when changed to Cys are capable of forming disulfide cross-links in the protein: 140/225, 136/222, and 136/225. These disulfides are able to form without addition of the Cu(phen)3(2+) oxidant. Similar to the 140-222 cross-link, none of the additional disulfides affect the spectral properties of rhodopsin. Also like the 140-222 bond, the 136-222 disulfide completely and reversibly inactivates the light-dependent activation of transducin by the receptor. In contrast, the 140-225 and 136-225 disulfides have no effect on the ability of rhodopsin to activate transducin. The pattern of cross-linking observed in Cys and disulfide scans of the protein is consistent with helical secondary structure in TM3 from 130 to 142 and in TM5 from 218 to 225.

Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1

“…Position 120, which we expect is buried in the membrane, forms a disulfide partially in the presence of CuP. Disulfide formation in a low dielectric environment suggests that position 120 is probably very near its molecular symmetry partner (14,15).…”

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

“…Disulfide cross-linking approaches have been used previously to investigate light-induced structural changes in the photoreceptor, rhodopsin (9,(25)(26)(27)(28)(29). However, this is the first study demonstrating the usefulness of a disulfide mapping strategy to investigate activity-dependent structural changes in a hormone-activated GPCR.…”

“…To address this issue, we decided to employ Cys substitution mutagenesis followed by disulfide cross-linking of Cys residues that are adjacent to each other in the three-dimensional structure of the receptor. This approach has been used successfully in the past to monitor dynamic changes in a number of different membrane proteins including various bacterial chemoreceptors (21-24) and, more recently, bovine rhodopsin (9,(25)(26)(27)(28)(29).To examine the potential proximity of the cytoplasmic ends of TM III and TM VI, we recently described a cross-linking protocol involving the use of a series of Cys-substituted mutant M 3 muscarinic receptors (30). The observed disulfide crosslinking patterns suggested that the cytoplasmic surface of the M 3 receptor protein is highly dynamic.…”

Structure of TRPV1 channel revealed by electron cryomicroscopy

“…Insoluble membranes were removed by centrifugation at 100,000 ϫ g for 1 h, using the Type 45 Ti rotor (Beckman Coulter). The supernatant containing TRPV1 was added to an affinity column of CNBr-activated Sepharose 4B coupled to 1D4 antibody (2 ml, 3-4 mg of Ab/ml) (42), and after washing with 20 mM sodium phosphate buffer, 500 mM NaCl, 1 M DDT, 10% glycerol, and 0.1% DM, protein was eluted by adding 0.1 mg/ml of 1D4 peptide into the washing buffer. Purified protein was resolved by electrophoresis on a 4 -20% linear gradient SDS-polyacrylamide gel (Invitrogen) and transferred to a supported nitrocellulose membrane.…”