A historical perspective on the lateral diffusion model of GTPase activation and related coupling of membrane signaling proteins.

Aspects of our discovery of lateral diffusion of the G protein coupled receptor (GPCR) rhodopsin and that a single activated rhodopsin can non-covalently catalyze GTP binding to thousands of GTPases per second on rod disk membranes via this diffusion are summarized herein. Rapid GTPase coupling to membrane-bound phosphodiesterase (PDE) further amplifies the signal via cGMP hydrolysis, essential to visual transduction. Important generalizations from this work are that biomembranes can uniquely concentrate, orient for reaction and provide a solvent appropriate to rapid, powerful and appropriately controlled sequential interaction of signaling proteins.

Multiple steps of phosphorylation of activated rhodopsin can account for the reproducibility of vertebrate rod single-photon responses.

Single-photon responses (SPRs) in vertebrate rods are considerably less variable than expected if isomerized rhodopsin (R*) inactivated in a single, memoryless step, and no other variability-reducing mechanisms were available. We present a new stochastic model, the core of which is the successive ratcheting down of R* activity, and a concomitant increase in the probability of quenching of R* by arrestin (Arr), with each phosphorylation of R* (Gibson, S.K.

G protein-coupled receptors as direct targets of inhaled anesthetics.

The molecular pharmacology of inhalational anesthetics remains poorly understood. Despite accumulating evidence suggesting that neuronal membrane proteins are potential targets of inhaled anesthetics, most currently favored membrane protein targets lack any direct evidence for anesthetic binding. We report herein the location of the binding site for the inhaled anesthetic halothane at the amino acid residue level of resolution in the ligand binding cavity in a prototypical G protein-coupled receptor, bovine rhodopsin.

Rapid irreversible G protein alpha subunit misfolding due to intramolecular kinetic bottleneck that precedes Mg2+ "lock" after GTP/GDP exchange.

Stoichiometric exchange of GTP for GDP on heterotrimeric G protein alpha (Galpha) subunits is essential to most hormone and neurotransmitter initiated signal transduction. Galphas are stably activated in a Mg2+ complex with GTPgammaS, a nonhydrolyzable GTP analogue that is reported to bind Galpha, with very high affinity. Yet, it is common to find that substantial amounts (30-90%) of purified G proteins cannot be activated.

Predictability of weak binding from X-ray crystallography: inhaled anesthetics and myoglobin.

Xenon and dichloromethane are inhalational anesthetic agents whose binding to myoglobin has been demonstrated by X-ray crystallography. We explore the thermodynamic significance of such binding using differential scanning calorimetry, circular dichroism spectroscopy, and hydrogen-tritium exchange measurements to study the effect of these agents on myoglobin folding stability. Though specific binding of these anesthetics might be expected to stabilize myoglobin against unfolding, dichloromethane actually destabilized myoglobin at all examined concentrations of this anesthetic (15, 40, and 200 mM).

Cooperative binding of inhaled anesthetics and ATP to firefly luciferase.

Firefly luciferase is considered a reasonable model of in vivo anesthetic targets despite being destabilized by anesthetics, as reflected by differential scanning calorimetry (DSC). We examined the interaction between two inhaled anesthetics, ATP, luciferase, and temperature, using amide hydrogen exchange, tryptophan fluorescence, and photolabeling in an attempt to examine this apparent discrepancy. In the absence of ATP/Mg2+, halothane and bromoform cause destabilization, as measured by hydrogen exchange, suggesting nonspecific interactions.

Halothane binding to a G protein coupled receptor in retinal membranes by photoaffinity labeling.

General anesthetics have been reported to alter the functions of G protein coupled receptor (GPCR) signaling systems. To determine whether these effects might be mediated by direct binding interactions with the GPCR or its associated G protein, we studied the binding character of halothane on mammalian rhodopsin, structurally the best understood GPCR, by using direct photoaffinity labeling with [(14)C]halothane. In the bleached bovine rod disk membranes (RDM), opsin and membrane lipids were dominantly photolabeled with [(14)C]halothane, but none of the three G protein subunits were labeled.

Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin.

Reduced effector activity and binding of arrestin are widely accepted consequences of GPCR phosphorylation. However, the effect of receptor multiphosphorylation on G protein activation and arrestin binding parameters has not previously been quantitatively examined. We have found receptor phosphorylation to alter both G protein and arrestin binding constants for light-activated rhodopsin in proportion to phosphorylation stoichiometry.

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect.

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 degrees C and pHs from 6.

Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin.

Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.

Volatile anesthetics alter protein stability.

1. We have used differential scanning calorimetry to measure the halothane induced change in stability of five lipid-free proteins in aqueous solution. 2.

Phosphorylation stabilizes the active conformation of rhodopsin.

Deactivation of many G protein coupled receptors (GPCRs) is now known to require phosphorylation of the activated receptor. The first such GPCR so analyzed was rhodopsin, which upon light activation forms an intramolecular equilibrium between the two conformers, metarhodopsin I and II (MI and MII). In this study, we find surprisingly that rhodopsin phosphorylation increases rather than diminishes the formation of MII, the conformation that activates G protein.

Conformational effects of calcium release from parvalbumin: comparison of computational simulations with spectroscopic investigations.

The effect of Ca2+ binding to parvalbumin was monitored by probes of conformation including absorption, fluorescence, circular dichroism (CD), infrared (IR) spectroscopy and differential scanning calorimetry. These experimental studies were compared with molecular dynamics computations on the structures of the Ca-bound and Ca-free forms of cod parvalbumin. The UV CD spectra show that removal of calcium results in a decrease in the alpha-helical content of the protein.

De novo design of native proteins: characterization of proteins intended to fold into antiparallel, rop-like, four-helix bundles.

The de novo design and characterization of a series of 51-residue helix-turn-helix peptides intended to dimerize into antiparallel four-stranded coiled coils is described. The sequence is based on a coiled coil heptad repeat Ncap-(Aa Zb Zc Ld Ze Zf Zg)3-turn- (Xa Zb Zc Ld Ze Zf Zg)3-Ccap-CONH2, where X is either Val or Ala. The overall topology was intended to be similar to that found in the Escherichia coli protein ROP.

Secondary recurrent carotid stenosis.

Purpose: Recurrent carotid stenosis after carotid endarterectomy has been extensively reported. The occurrence, however, of another ipsilateral restenosis that requires a third carotid operation is rare. The purpose of this study was to evaluate possible risk factors and the most efficacious management of the patient with "secondary" recurrent carotid stenosis.

Effect of photoregeneration on the calculation of the amount of rhodopsin bleached by small flashes.

A sample of rhodopsin that is exposed to a series of small light flashes of equal intensity is expected to bleach in successively smaller decrements in proportion to the remaining unbleached rhodopsin. The exponential depletion law describing this effect has been used as a rapid, convenient, and intuitive method for determining the fraction of rhodopsin bleached per flash. This method is commonly assumed to be free of error provided the amount bleached is small, so that there is no significant photoregeneration.

Identification of a G-protein in depolarizing rod bipolar cells.

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin.

Tubulin in bovine retinal rod outer segments.

Bovine rod outer segment (ROS) preparations contain a major 58 kDa protein doublet that was identified by immunoblot as tubulin. Quantification by gel densitometry showed that the total amount of tubulin was 5- to 10-fold higher than that attributable to the rod axoneme, suggesting additional role(s) for tubulin in photoreceptor cells. Approximately 20% of this nonaxonemal tubulin (15% of total tubulin) is tightly associated with outer segment membranes.

The effect of GDP on rod outer segment G-protein interactions.

The role of GDP has heretofore been little studied in the analysis of visual receptor G-protein (G) interactions. Here we use kinetically resolved absorption and light scattering spectroscopy, centrifugation, porous membrane filtration, and enzyme assay to compare the effectiveness of GDP with that of GTP or gamma-thio-guanosine-5'-triphosphate in the modulation of G-protein binding to rod disc membranes and activated receptor (R*). We also compare effectiveness of GDP with that of GTP in the separation of G alpha and G beta gamma subunits and in activation of effector, cGMP phosphodiesterase.

cGMP-dependent cation channel of retinal rod outer segments.

Light-modulated cytoplasmic cGMP simultaneously controls plasma membrane Na+ conductance in visual excitation and Ca2+ entry into rods by direct interaction with the cation channel. Cytoplasmic Ca2+ in turn may set operating points and contribute to the dynamics of several enzymes that regulate cGMP levels in the dark, recovery from excitation and receptor adaptation or down regulation. Similar channels may couple electrical activity to internal nucleotide metabolism in other tissues.