Abstract
The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cis-retinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr(251) in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.
Highlights
Based on the proposed existence of a common activation mechanism, which has been correlated with the presence of a set of conserved residues among the G-protein-coupled receptor (GPCR) superfamily, nomenclature has been established for an easy comparison among their amino acid sequences [11]
Mutations E134Q, E247A, E247Q, E134Q/E247A, and E134Q/E247Q—These mutants were expressed to an extent similar to that of WT rhodopsin according to their absorbance spectra (Fig. 2, A and C)
E247A and E247Q showed a chromophore formation similar to WT rhodopsin, whereas E134Q and E134Q/E247A showed some differences judged from the A280/A500 ratios (Table 2)
Summary
Materials—All buffers and chemicals were purchased from Panreac and Sigma. Oligonucleotides were obtained from Operon (Qiagen). The cells were incubated with 20 M 11-cisretinal for 5 h for reconstitution, solubilized in buffer B for 1 h, and centrifuged at 35,000 rpm for 35 min. All these procedures were carried out at 4 °C. The protein was placed in a box containing a mixture of DPPC lipids and water molecules generated and equilibrated according to the procedure described previously [41]. The box contained 256 lipids and ϳ17,000 water molecules. The pressure in the three coordinate directions was kept at 0.1 MPa. Single mutants were constructed using cassette mutagenesis, and double mutants were obtained by PCR
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