We have found that the 48-kDa protein (or S-antigen 48k) of the rod photoreceptor enhances the light-induced formation of the photoproduct metarhodopsin II (MII) from prephosphorylated rhodopsin. The effect is analogous to the known enhancement of MII (extra-MII) that results from selective interaction of MII with G-protein. We have determined some parameters of the MII-48k interaction by measuring the extra-MII absorption change induced by the 48-kDa protein. The amplitude saturation yields a dissociation constant for the MII-48k complex on the order of 50 nM. At the technical limit of these measurements, 13.7 degrees C and 12 microM 48-kDa protein, we find a rate of 2.3 s-1 for formation of the 48k-MII complex. Extrapolation of these values to cellular conditions yields an occupation time of phosphorylated MII by 48k less than 200 ms. This is short compared to estimated rates of phosphorylation. The temperature dependence of the MII-48k formation rate is very high (Q10 for 5 degrees C/15 degrees C = 9-10). The related Arrhenius activation energy (165 kJ mol-1) is correspondingly high and indicates a considerable transient chemical change during the binding process.

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.

Molecular dynamics of a main chain thermotropic liquid crystal polymer in the smectic A phase has been investigated using multipulse dynamic nuclear magnetic resonance (NMR) techniques. Transverse deuteron spin relaxation times T~; from quadrupole echo pulse trains (modified Carr-Purcell-Meiboom-Gill sequence) measured for deuterons in the aromatic rings of the mesogenic units are obtained as a function of pulse spacing 7 sample orientation eN' and temperature. Just below the. nematic-smectic A phase transition, the relaxation times exhibit a linear dispersion regime ~; -7-1 consistent with smectic director fluctuations. At lower temperatures, the dispersion step gradually disappears, indicating that faster molecular motions are the dominant transverse relaxation process. The observed anisotropy in ~;, measured at short pulse spacings, approximately follows the (sin 4 e N) -I dependence expected for axial diffusion in a highly ordered medium. Analysis of the experiments is achieved employing a density operator treatment based on the stochastic Liouville equation. The intramolecular motion is identified with phenyl ring flips and is the fastest process studied, with correlation times varying from 10-10 to 10-7 s over the temperature range investigated. Intermolecular (individual molecule) dynamics are somewhat slower and have been interpreted as rotational diffusion in an orienting potential. The correlation times for intermolecular motion exhibit non-Arrhenius behavior approaching the glass transition, following a temperature dependence described by the Williams-Landel-Ferry equation over six orders of magnitude. This result indicates a strong coupling of the intermolecular motion to the glass transition process. The slowest motion affecting transverse deuteron spin relaxation is assigned to smectic director fluctuations or undulation waves. Analysis of the ~; dispersion yields information concerning the viscoelastic properties of the polymer. At T=418 K, a splay elastic constant of KI =2X lO-11 N has been estimated. Using the experimentally accessible value for the long wavelength cutoff of the elastic modes, the root mean square fluctuation 1/2 of the director is calculated to be 4°.

The protein-detergent interaction in rhodopsin-detergent micelles has been investigated by using formation of metarhodopsin II (MII) as a monitor. Two detergents of different structural rigidity have been applied. One of them is [3-(lauroyloxy)propyl]phosphorylcholine, which has a high conformational flexibility in its hydrophobic moiety like most of the known detergents for rhodopsin. This deoxylysolecithin was originally designed as a detergent for membrane proteins by Weltzien [Weltzien, H. U. (1979) Biochim. Biophys. Acta 559, 259-287]. The other detergent, which is highly rigid in its hydrophobic part, has been developed for this study. It consists of a biphenyl derivative and a hydrophilic octaethylene oxide group. Both the formation kinetics of MII and the position of its equilibrium with its tautomeric form, metarhodopsin I (MI), strongly differed in the deoxylysolecithin and biphenyl detergent. Deoxylysolecithin caused very fast MII formation and shifted the equilibrium strongly to MII, like other detergents with alkyl chains as the hydrophobic part. In the biphenyl detergent, however, formation of MII was slow and the MI/MII equilibrium similar to that in the native system. For rhodopsin reconstituted in lipid bilayers, normal MII formation requires a well-adjusted fluidity of the hydrocarbon environment of the protein [Baldwin, P. A., & Hubbell, W. L. (1984) Biochemistry 24, 2633-2639], which was explained by an appropriate interfacial pressure at the protein-lipid interface. Extension of this concept would indicate that in the micellar core a degree of fluidity comparable to that of the disk membrane is just achieved with the highly rigid biphenyl structure.(ABSTRACT TRUNCATED AT 250 WORDS)

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