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Structural transitions of transmembrane helix 6 in the formation of metarhodopsin I.

ABSTRACT: Absorption of light by the visual pigment rhodopsin triggers a rapid cis-trans photoisomerization of its retinal chromophore and a series of conformational changes in both the retinal and protein. The largest structural change is an outward tilt of transmembrane helix H6 that increases the separation of the intracellular ends of H6 and H3 and opens up the G-protein binding site. In the dark state of rhodopsin, Glu247 at the intracellular end of H6 forms a salt bridge with Arg135 on H3 to tether H6 in an inactive conformation. The Arg135-Glu247 interaction is broken in the active state of the receptor, and Arg135 is then stabilized by interactions with Tyr223, Met257, and Tyr306 on helices H5, H6, and H7, respectively. To address the mechanism of H6 motion, solid-state NMR measurements are undertaken of Metarhodopsin I (Meta I), the intermediate preceding the active Metarhodopsin II (Meta II) state of the receptor. (13)C NMR dipolar recoupling measurements reveal an interhelical contact of (13)C?-Arg135 with (13)C?-Met257 in Meta I but not with (13)C?-Tyr223 or (13)C?-Tyr306. These observations suggest that helix H6 has rotated in the formation of Meta I but that structural changes involving helices H5 and H7 have not yet occurred. Together, our results provide insights into the sequence of events leading up to the outward motion of H6, a hallmark of G protein-coupled receptor activation.

SUBMITTER: Eilers M

PROVIDER: S-EPMC3428503 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Structural transitions of transmembrane helix 6 in the formation of metarhodopsin I.

Eilers Markus M Goncalves Joseph A JA Ahuja Shivani S Kirkup Colleen C Hirshfeld Amiram A Simmerling Carlos C Reeves Philip J PJ Sheves Mordechai M Smith Steven O SO

The journal of physical chemistry. B 20120517 35

Absorption of light by the visual pigment rhodopsin triggers a rapid cis-trans photoisomerization of its retinal chromophore and a series of conformational changes in both the retinal and protein. The largest structural change is an outward tilt of transmembrane helix H6 that increases the separation of the intracellular ends of H6 and H3 and opens up the G-protein binding site. In the dark state of rhodopsin, Glu247 at the intracellular end of H6 forms a salt bridge with Arg135 on H3 to tether ...[more]

PMID: 22564141

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Project description:We have previously studied the unfolding equilibrium of bacterioopsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterioopsin perturbed by increasing ethanol concentrations [Nannepaga et al. (2004) Biochemistry 43, 50-59]. We have further investigated this transition and find that the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterioopsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are 5.46 for residues 72-248 (Cys 163) and 7.36 for residues 1-166 (Lys 41). When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding in the range of -0.54 to -3.5 kcal/mol. The results suggest that the C-terminal helix of bacterioopsin is less stably folded than the N-terminal helices. We analyzed the pairwise helix-helix interaction surfaces of bacteriorhodopsin and three other seven-transmembrane-helix proteins on the basis of crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl-terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high-resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helix-helix interactions.

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Structural transitions of transmembrane helix 6 in the formation of metarhodopsin I.

Publications

Structural transitions of transmembrane helix 6 in the formation of metarhodopsin I.

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