The recent report of the crystal structure of rhodopsin provides insights concerning structure-activity relationships in visual pigments and related G protein-coupled receptors (GPCRs). The seven transmembrane helices of rhodopsin are interrupted or kinked at multiple sites. An extensive network of interhelical interactions stabilizes the ground state of the receptor. The ligand-binding pocket of rhodopsin is remarkably compact, and several chromophore-protein interactions were not predicted from mutagenesis or spectroscopic studies. The helix movement model of receptor activation, which likely applies to all GPCRs of the rhodopsin family, is supported by several structural elements that suggest how light-induced conformational changes in the ligand-binding pocket are transmitted to the cytoplasmic surface. The cytoplasmic domain of the receptor includes a helical domain extending from the seventh transmembrane segment parallel to the bilayer surface. The cytoplasmic surface appears to be approximately large enough to bind to the transducin heterotrimer in a one-to-one complex. The structural basis for several unique biophysical properties of rhodopsin, including its extremely low dark noise level and high quantum efficiency, can now be addressed using a combination of structural biology and various spectroscopic methods. Future high-resolution structural studies of rhodopsin and other GPCRs will form the basis to elucidate the detailed molecular mechanism of GPCR-mediated signal transduction.

The binding constants, K(1) and K(2), and the number of Ca(2+) ions in each of the two high affinity sites of Ca(2+)-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K(1) and K(2), it was found that two hydrogen ions are released for each Ca(2+) bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K(s), for the binding of Ca(2+) to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K(s) and its pH dependence with those of K(1) and K(2) showed that the site corresponding to K(s) is to be identified with that of K(2). This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca(2+) that causes the blue to purple color change.Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.

Bacteriorhodopsin contains Ca2+ and Mg2+ ions whose removal inhibits its proton pump function. The binding constants of Ca2+ to the high-affinity sites were determined by the use of a calcium ion specific electrode. The unavailability of magnesium ion specific electrode prevented a similar determination for Mg2+. The binding constant of Mg2+ to the binding site of highest affinity is determined by using a calcium ion selective electrode to measure the concentration of free Ca2+ in competition with Mg2+ for the binding. The binding constant of Mg2+ to the second high affinity site is determined spectrally. The two high-affinity binding constants for Mg2+ are compared with those obtained for Ca2+ in the absence and the presence of Mg2+. The fact that the presence of low concentration of one metal ion does not affect the binding constant of the other metal ion to the other binding site supports the assumption of the independence of the two high-affinity sites of one another. The difference in the observed values of the binding constants of the two high-affinity sites for Ca2+ and Mg2+ is qualitatively discussed in terms of the enthalpy and entropy changes in the binding equilibrium.

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