John H. Parkes

Temperature and pH dependence of the metarhodopsin I-metarhodopsin II equilibrium and the binding of metarhodopsin II to G protein in rod disk membranes.

The equilibria between metarhodopsins I and II (MI and MII) and the binding of MII to retinal G protein (G) were investigated, using the dual wavelength absorbance response of rod disk membrane (RDM) suspensions to a series of small bleaches, together with a nonlinear least-squares fitting procedure that decouples the two reactions. This method has been subjected to a variety of theoretical and experimental tests that establish its validity. The two equilibrium constants, the amount of active G protein (that can bind to and stabilize MII) and the fraction bleached by the flash, have been determined without a priori assumptions about these values, at temperatures between 0 and 15 °C and pHs from 6.2 to 8.2. Binding of G to MII in normal RDM exhibits 1:1 stoichiometry ( not cooperative), relatively weak, 2 -4 × 104 M-1 affinity on the membrane, with a pH dependence maximal at pH 7.6, and a low thermal coefficient. The reported amount of active G remained constant even when its binding constant was reduced more than 10-fold at low pH. The method can readily be applied to the binding of MII to other proteins or polypeptides that stabilize its conformation as MII. It appears capable of determining many of the essential physical constants of G protein coupled receptor interaction with immediate signaling partners and the effect of perturbation of environmental parameters on these constants. Members of the G protein coupled receptor (GPCR) 1 family number in the thousands, functioning to transduce extracellular signals that cells depend on for most of their life processes. GPCRs are postulated to exist in several intramolecular conformations. It is thought that signals (agonists) activate them by stabilizing the particular receptor conformation that binds to and activates G proteins. Receptor-bound G proteins lose their GDP and stabilize the receptor in the agonist-activated conformation. This paradigm appears to be qualitatively supported by data from many receptor systems that show the midpoint of agonist binding curves to be shifted to lower agonist concentration in the presence of G protein (the agonist-receptor high-affinity state) [see Kenakin (1) and references therein]. When GTP binding subsequently releases the bound G protein, the receptor returns to its low agonist affinity state. The kinetics and thermodynamics of receptor -G protein interactions have resisted quantitative analysis partly because these proteins are restricted to the surfaces of cellular membranes where they are less accessible to common biochemical techniques. Difficulty in obtaining natural or recombinant receptor and G proteins in amounts necessary for many experimental approaches has also slowed progress. Receptor -G protein interactions in rod disk membranes closely mirror those of other GPCRs. In its least active state, rhodopsin contains the inverse agonist and chromophore, 11 cis-retinal, which is covalently attached to the receptor protein. Light absorbed by rhodopsin can isomerize this chromophore toall-trans-retinal, the agonist of rhodopsin activation. Resultant protein charge redistribution and conformational changes produce rapid changes in rhodopsin’s visible light absorption spectra, ultimately forming a pH and temperature-dependent equilibrium between metarhodopsins I (MI, λmax 480 nm) and II (MII,λmax 387 nm) (2, 3). These spectroscopic properties of rhodopsin’s activation path intermediates, together with the natural abundance of rhodopsin and retinal G protein, make possible the quantitative investigation of GPCR signaling events that have been virtually impossible to study in other GPCR systems. MII is the first rhodopsin-bleaching intermediate able to bind the rod G protein, transducin ( 4), forming MII‚G, the visual homologue of the agonist-receptor high-affinity state. Binding to G stabilizes MII ( 5), removing it from the MIMII equilibrium and, in the absence of guanine nucleotides, † Supported by NIH Grants EY00012, EY01583, and EY07035. * To whom correspondence should be addressed. Phone: 215-8986917. Fax: 215-573-8093. E-mail: liebmanp@mail.med.upenn.edu. 1 Abbreviations: f, fraction of rhodopsin bleached per flash; G, G protein; GDP, guanosine 5 ′-diphosphate; GPCR, G protein coupled receptor; GTP, guanosine 5 ′-triphosphate; GTP γS, guanosine 5 ′-O-(3thio-triphosphate); Ka, MI-MII equilibrium constant;Kb, MII ‚G binding constant; MI, metarhodopsin I; MII, metarhodopsin II; MIII, metarhodopsin III; MOPS, 3-( N-morpholino)propanesulfonic acid; OD, optical density;∆OD, optical density difference; R, rhodopsin, R*, bleached rhodopsin; RDM, rod disk membranes; [R] mem, [R]sol, rhodopsin membrane phase or solution average concentration; sRDM, hypotonically stripped RDM; SSR, sum of squared residuals; usRDM, urea stripped RDM. 6862 Biochemistry1999,38, 6862-6878 10.1021/bi9827666 CCC:

Mechanism of cGMP control in retinal rod outer segments

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Circular dichroism of rhodopsins and porphyropsins.

Abstract Rapid progress has been made in understanding the important roles of intramembrane lateral diffusion, GTP-binding, protein-protein interaction and receptor phosphorylation in amplified PDE activation and cGMP control in retinal rod outer segments. At each investigative turn, there are close mechanistic similarities to GTP-dependent hormonal activation of adenylate cyclase and in many cases, published studies of the latter have guided work on the visual system. The abundance of controller proteins and ready applicability of quantitative methods such as spectroscopy, peripheral membrane protein elution, optical and pH kinetic recording have produced an advanced dynamic picture of how the four major integral and peripheral membrane proteins of RDM interact to rapidly control cyclic nucleotide levels.

Temperature and pH dependence of the metarhodopsin I-metarhodopsin II kinetics and equilibria in bovine rod disk membrane suspensions

Circular dichroism and absorption spectra were determined for digitonin extracts of three rhodopsins: cattle, grass frog, and pigeon; and three porphyropsins: channel catfish, bluegill sunfish, and redear sunfish. A comparison of these spectra shows the following: (1) Porphyropsins, like rhodopsins, exhibit two positive CD peaks in the spectral region 320--700 nm: an alpha peak at about 520 nm and a small beta peak at about 355 nm. These peaks substantially diminish upon bleaching. (2) In the CD spectra the alpha peaks of the porphyropsins are larger than the alpha peaks of the rhodopsins, while the beta peaks are smaller than those of the rhodopsins. This is just the opposite of the corresponding relationship between the peaks in the absorption spectra. (3) The maxima of these peaks in the CD spectra of rhodopsins and porphyropsins are consistently blueshifted from corresponding maxima in absorption spectra. (4) Some of the visual pigments show additional positive CD peaks in the spectral region 250--320 nm. In all the visual pigments studied, the CD spectra in this region decrease on bleaching. No reciprocal relationship is observed between any of the CD bands in the visible and near ultraviolet region of the spectrum.