Kinetic study on the equilibrium between membrane-bound and free photoreceptor G-protein

Abstract

Formation of the complex between photoreceptor G-protein (G) and photoactivated rhodopsin (RM) leads to a change in the light scattering of the disk membranes (binding signal or signal P). The signal measured on isolated disks (so-called PD signal) is exactly stoichiometric in its final level to bound G-protein but its kinetics are much slower than the RMG binding reaction. In this study on isolated disks, recombined with G-protein, we analyzed the PD-signal level and kinetics as a function of flash intensity and compared it to the RMG-complex formation monitored spectroscopically (by extra metarhodopsin II). The basic observation is that the initial slopes of the PD signals decrease with flash intensity when the signals are normalized to the same final level. This finding prevents an explanation of the scattering signal by a slow postponed reaction of the RMG complex. We propose to interpret the scattering change as a redistribution of G-protein between a membrane-bound and a solved state. The process is driven by the complexation of membrane-bound G to flash-activated rhodopsin (RM). The experimental evidence for this two-state model is the following: The intensity dependence of the initial rate of the PD signal is explained by the model. Under the assumption of a bimolecular reaction of free G with sites at the membrane, equal to rhodopsin in their concentration, the measured rates yield a KD of 10(-5) M. Evaluation of the extra MII kinetics yields a biphasic rise at saturating flashes. The measured rates fit to the supply of free and membrane-bound G-protein for the reaction with RM. Quantitative estimation of the expected scattering intensity changes gives a comprehensive description of binding signal and dissociation signal by the gain and loss of G-protein scattering mass. The temperature dependence of the PD-signal rate leads to an activation energy of the membrane-association process of E alpha = 44 kJ/mol.

Maximal Rate and Nucleotide Dependence of Rhodopsin-catalyzed Transducin Activation

“…2B, trace a). Accordingly, dissociation of G t mb from the membrane upon activation is seen as decrease of LS ("dissociation signal" (20,36); Fig. 2B, trace b).…”

“…Notably, binding signals are generally slow as compared with dissociation signals. Previous work resolved this apparent conflict by the finding that, although binding of G t mb to R* is fast, interaction of G t sol with the membrane is slow (36). Consequently, the activation rate of G t sol is limited by its membrane binding, thereby leading to artificially slowed activation rates when total active G t * formation is assayed under conditions where a significant fraction of G t GDP is solubilized.…”

“…We thus used the kinetic light-scattering technique to obtain initial rates of G t mb activation. Previous work established the dissociation signal as a real time monitor of the fast activation of the membrane-bound fraction of G t GDP (20,36,37). A precise calibration can be obtained from the dependence on [R*] (37, 38) and/or from classical biochemical assays (this work).…”

Signal transfer from rhodopsin to the G-protein: Evidence for a two-site sequential fit mechanism

“…The changes in a dissociation signal were recorded as before (25,26). Measuring conditions were 3 M rhodopsin (WM), 0.4 M Gt, 500 M GTP, 20°C; 0.9% of rhodopsin was flash-activated.…”

Arrestin and Its Splice Variant Arr1–370A(p44)

“…In the present study, the binding signals are used as a tool to determine the state of membrane binding (prior to the flash) of a protein. The shift of protein mass from solution to the membrane becomes the larger the less of the protein that is bound to the membrane before receptor activation (30,42). No binding signal will be seen when the protein is completely bound to the membrane.…”