AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor.

The bicyclam AMD3100 (formula weight 830) blocks HIV-1 entry and membrane fusion via the CXCR4 co-receptor, but not via CCR5. AMD3100 prevents monoclonal antibody 12G5 from binding to CXCR4, but has no effect on binding of monoclonal antibody 2D7 to CCR5. It also inhibits binding of the CXC-chemokine, SDF-1alpha, to CXCR4 and subsequent signal transduction, but does not itself cause signaling and has no effect on RANTES signaling via CCR5. Thus, AMD3100 prevents CXCR4 functioning as both a HIV-1 co-receptor and a CXC-chemokine receptor. Development of small molecule inhibitors of HIV-1 entry is feasible.

Rhodopsin mutants that bind but fail to activate transducin.

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.

How color visual pigments are tuned.

The absorption maximum of the retinal chromophore in color visual pigments is tuned by interactions with the protein (opsin) to which it is bound. Recent advances in the expression of rhodopsin-like transmembrane receptors and in spectroscopic techniques have allowed us to measure resonance Raman vibrational spectra of the retinal chromophore in recombinant visual pigments to examine the molecular basis of this spectral tuning. The dominant physical mechanism responsible for the opsin shift in color vision is the interaction of dipolar amino acid residues with the ground- and excited-state charge distributions of the chromophore.

Rhodopsin: a prototypical G protein-coupled receptor.

A variety of spectroscopic and biochemical studies of recombinant site-directed mutants of rhodopsin and related visual pigments have been reported over the past 9 years. These studies have elucidated key structural elements common to visual pigments. In addition, systematic analysis of the chromophore-binding pocket in rhodopsin and cone pigments has led to an improved understanding of the mechanism of the opsin shift, and of particular molecular determinants underlying color vision in humans. Identification of the conformational changes that occur on rhodopsin photoactivation has been of particular recent concern. Assignments of light-dependent molecular alterations to specific regions of the chromophore have also been attempted by studying native opsins regenerated with synthetic retinal analogs. Site-directed mutagenesis of rhodopsin has also provided useful information about the retinal-binding pocket and the molecular mechanism of rhodopsin photoactivation. Individual molecular groups have been identified to undergo structural alterations or environmental changes during photoactivation. Analysis of particular mutant pigments in which specific groups are locked into their respective "off" or "on" states has provided a framework to identify determinants of the active conformation, as well as the minimal number of intramolecular transitions required to switch between inactive and active conformations. A simple model for the active state of rhodopsin can be compared to structural models of its ground state to localize chromophore-protein interactions that may be important in the photoactivation mechanism. This review focuses on the recent functional characterization of site-directed mutants of bovine rhodopsin and some cone pigments. In addition, an attempt is made to reconcile previous key findings and existing structural models with information gained from the analysis of site-directed mutant pigments.

A differential labelling model for determining the number of catalytically essential carboxyl groups in fumarase.

We have developed a mathematical model of the nonideal case in which enzymatic activity changes may also result from modification of non-essential groups. As an illustration of this method, the number of essential carboxyl groups in pig heart fumarase (L-malate hydro-lyase, ED 4.2.1.2) was determined by the differential labeling technique. Enzymatic activity was related to the number of modified carboxyl groups according to the model and the results were compatible with the existence of two essential carboxyl groups in fumarase.

The steric trigger in rhodopsin activation.

Rhodopsin is the seven transmembrane helix receptor responsible for dim light vision in vertebrate rod cells. The protein has structural homology with the other G protein-coupled receptors, which suggests that the tertiary structures and activation mechanisms are likely to be similar. However, rhodopsin is unique in several respects. The most striking is the fact that the receptor "ligand", 11-cis retinal, is covalently bound to the protein and is converted from an "antagonist" to an "agonist" upon absorption of light. NMR studies of rhodopsin and its primary photoproduct, bathorhodopsin, have generated structural constraints that enabled docking of the 11-cis and all-trans retinal chromophores into a low-resolution model of the protein proposed by Baldwin. These studies also suggest a mechanism for how retinal isomerization leads to rhodopsin activation. More recently, mutagenesis studies have extended these results by showing how the selectivity of the retinal-binding site can be modified to favor the all-trans over the 11-cis isomer. The structural constraints produced from these studies, when placed in the context of a high-resolution model of the protein, provide a coherent picture of the activation mechanism, which we show involves a direct steric interaction between the retinal chromophore and transmembrane helix 3 in the region of Gly121.

Evidence for the specific interaction of a lipid molecule with rhodopsin which is altered in the transition to the active state metarhodopsin II.

Comparing the FTIR difference spectra of the rhodopsin --> metarhodopsin II transition in membranes and in dodecylmaltoside detergent, characteristic variations are observed between 1715 and 1750 cm(-1). By repeating the measurements with the rhodopsin mutant D83N/E122Q, the spectral variation between the samples in membranes versus detergent could be assigned to a difference band at 1743(+)/1724(-) cm(-1), which does not exhibit a deuteration-induced downshift. We provide evidence that this band is probably caused by the C=O stretch of only one ester group of one lipid molecule. This group interacts with the dark state of rhodopsin, whereas in metarhodopsin II, the lipid molecule behaves as if it were in the bulk lipid phase.

Mutation of a conserved cysteine in the X-linked cone opsins causes color vision deficiencies by disrupting protein folding and stability.

PURPOSE: To test the effects of disruption of a conserved cysteine in the green cone opsin molecule on light-activated isomerization, transducin activation, folding, transport, and protein half-life. METHODS: Stable cell lines were established by transfecting 293-EBNA cells with a plasmid containing wild-type or mutant (C203R, C203S, C126S, C126S/C203S) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of cadmium chloride and analyzed by spectra, transducin activation, Western blotting, pulse-labeling with immunoprecipitation, and immunocytochemistry. RESULTS: The C203R mutation disrupts the folding and half-life of the green opsin molecule and its abilities to absorb light at the appropriate wavelength and to activate transducin. Similar disruption of folding, half-life, and light activation occurs when Cys203 or its presumed partner for formation of a disulfide bond (Cys126) is replaced by serine residues. CONCLUSIONS: Like rhodopsin, the folding of the cone opsins appears to be dependent on the formation of a disulfide bond between the third transmembrane helix and the second extracellular loop. Disruption of this disulfide bond represents a cause of color vision deficiencies that is unrelated to spectral shifts of the photopigment.

Colour tuning mechanisms of visual pigments.

Spectral tuning by visual pigments involves modulation of physical properties of the 11-cis-retinylidene protonated Schiff base (PSB) chromophore by amino acid side chains in and around the chromophore-binding pocket. Specific molecular contacts between the chromophore and the amino acid side chains of the opsin chromophore-binding pocket have been determined recently using an interdisciplinary approach consisting of site-directed mutagenesis, optical and vibrational spectroscopy, and molecular graphics modelling. These studies provide insight into the mechanism of spectral tuning among visual pigments. In blue pigments a majority of the opsin shift is caused by polar amino acid side chains arrayed about the PSB to increase the energy gap between the ground (S0) and excited states (S1). In addition, a specific tyrosine near the chromophore ring causes a decrease in solvent polarizability. Other amino acid residues alter the binding pocket structure to strengthen electrostatic interaction between the PSB and its counterion and/or solvent dipoles. In the green and red pigments, the work of Kochendoerfer et al (1997; Biochemistry 26:6577-6587) demonstrates that local structural perturbations at the PSB or elsewhere are not responsible for spectral tuning. Instead, the green-to-red opsin shift is best explained by dipolar side chains near the chromophore ring that lower the transition energy that occurs upon electronic excitation by affecting the change in electric dipole moment. In summary, the absorption maximum of a visual pigment is primarily regulated by the interaction of the chromophore charge distribution with dipolar residues in its opsin chromophore-binding pocket. The work presented in this paper is reported in greater detail in Lin et al.

The traveler's medical kit.

This article presents a comprehensive list of drugs and other medical supplies that may be of use to travelers for treating common infections and noninfectious travel-related illness. Recommendations are offered for using the medications to treat specific common medical problems while traveling. Sample medical kits, the contents of which vary depending on itinerary and length of stay, are presented.

Photoactivated state of rhodopsin and how it can form.

A variety of spectroscopic and biochemical studies of the photoreceptor rhodopsin have revealed conformation changes which occur upon its photoactivation. Assignment of these molecular alterations to specific regions in the receptor has been attempted by studying native opsin regenerated with synthetic retinal analogs or recombinant opsins regenerated with 11-cis retinal. We propose a model for the photoactivation mechanism which defines 'off' and 'on' states for individual molecular groups. These groups have been identified to undergo structural alterations during photoactivation. Analysis of mutant pigments in which specific groups are locked into their respective 'on' or 'off' states provides a framework to identify determinants of the active conformation as well as the minimal number of intramolecular transitions to switch to this conformation. The simple model proposed for the active-state of rhodopsin can be compared to structural models of its ground-state to localize chromophore-protein interactions that may be important in the photoactivation mechanism.

Total synthesis and expression of a gene for the alpha-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin).

To facilitate structure-function studies by site-specific mutagenesis, we have synthesized a gene for the alpha-subunit of the bovine rod outer segment (ROS) guanine nucleotide-binding protein (transducin). The gene codes for the native amino acid sequence and contains, by design, 38 unique restriction sites which are uniformly spaced. This enables mutagenesis in any part of the gene by restriction fragment replacement. The gene is 1076 base pairs in length. It was constructed from 44 synthetic oligonucleotides which were joined enzymatically in vitro into four fragments which were cloned. The synthetic transducin gene and cDNA encoding transducin were expressed to similar levels in monkey kidney cells (COS-1) using a vector in which transcription was under the control of the adenovirus major late promoter.

Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state.

The difference of rhodopsin and metarhodopsin II (MII) absorption spectra exhibits a characteristic pattern in the UV wavelength range, consisting of peaks at 278, 286, 294, 302 nm. These difference bands are thought to result from the perturbation of the environments of tryptophan and/or tyrosine residues. We used site-directed mutagenesis to investigate the contribution of tryptophan absorption to these spectral features. Each of the five tryptophan residues in bovine rhodopsin was replaced by either a phenylalanine or a tyrosine. The mutant pigments (W35F, W126F, W161F, W175F, W265F/Y) were prepared and studied by UV-visible photobleaching difference spectroscopy. The difference spectra of the W35F and W175F mutants were identical to that of rhodopsin, whereas in the W161F mutant, the magnitudes of the 294- and 302-nm bands were slightly lowered. The differential absorbance at 294 nm was reduced by over 50% in the W126F and W265F/Y mutants. The difference peak at 302 nm was reduced in the W265F/Y mutants, but was almost completely absent in the W126F mutant. These data indicate that the difference bands at 294 and 302 nm originate from the perturbations of Trp126 and Trp265 environments resulting from a general conformational change concomitant with MII formation and receptor activation. Model studies on tryptophan absorption indicate that the difference peak at 294 nm is due to the differential shift of the Lb absorption of the indole side chain as a result of decreased hydrophobicity or polarizability of the Trp126 and Trp265 environments. The resolution of the 302-nm band, assigned to the differential shift of the indole La absorption, is consistent with hydrogen-bonding interactions of the indole N-H groups of Trp126 and Trp265 becoming weaker in MII. These results suggest that the photoactivation of rhodopsin involves a change in the relative disposition of transmembrane helices 3 and 6, which contain Trp126 and Trp265 respectively, within the alpha-helical bundle of the receptor.

Light-dependent transducin activation by an ultraviolet-absorbing rhodopsin mutant.

The photoactivation pathway of an ultraviolet-absorbing rhodopsin mutant was studied. The mutant pigment, in which the retinylidene Schiff base counterion, Glu113, was replaced by glutamine (E113Q), was known to exist in a pH-dependent equilibrium between spectral forms absorbing at about 380 and 490 nm. The 380-nm form contains an unprotonated Schiff base chromophore linkage, whereas the 490-nm form contains a protonated Schiff base chromophore linkage. The role of the Schiff base proton in photoactivation was investigated by measuring transducin activation as a function of photoactivation wavelength. The transducin activation action spectra of rhodopsin and of mutant E113Q were found to be very similar to their UV-visible absorption spectra. Thus, the 380-nm UV form of the mutant E113Q could be activated directly by UV light to catalyze nucleotide exchange by transducin. The quantum efficiency of photoactivation of the UV-absorbing form of E113Q was similar to that of its visible-absorbing form. These results show that the presence of a protonated Schiff base in the ground state is not necessarily required for efficient photoactivation of visual pigments. They support the hypothesis that the key role of the protonated Schiff base in visible-absorbing pigments is to stabilize the ground state and to allow absorbance at wavelengths above about 420 nm. The findings are also consistent with transducin activation studies of mutant apoproteins regenerated with all-trans-retinal, or of mutant apoproteins alone, suggesting that the active state of rhodopsin can be formed via a number of pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the rhodopsin-transducin interaction by a highly conserved carboxylic acid group.

Rhodopsin is a member of a family of G protein-coupled receptors which share structural and functional homologies. A tripeptide sequence (Glu or Asp/Arg/Tyr) at the cytoplasmic border of the third transmembrane segment is conserved among most of these receptors. This region is involved in G protein activation in rhodopsin as well as in other receptors. The role of the conserved Glu-134 was studied by site-specific mutagenesis of rhodopsin in combination with a real-time fluorescence assay of G protein (transducin) activation. Assay conditions were chosen under which the transducin activation rate was determined either by rhodopsin-transducin complex formation or by GTP gamma S-induced complex dissociation. Glu-134 was replaced by Gln in order to mimic the protonated state of the carboxylic acid group. This mutation caused the pH dependency of complex formation to extend to the alkaline range as compared with rhodopsin. Replacement of Glu-134 by Asp had an opposite but less pronounced effect on the pH dependency and lowered the overall efficiency of transducin activation. The acidity constant (pKa) of the residue at position 134 did not directly determine the pH sensitivity of complex formation, indicating that other amino acid residues contribute to a titratable binding domain that includes Glu-134. In contrast, the pH sensitivity of GTP gamma S-induced complex dissociation was not changed by the mutations, although absolute rates were affected. The data suggest that the protonated state of Glu-134 favors binding of rhodopsin to transducin and that Glu-134 is not titratable in the rhodopsin-transducin complex.

Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin.

The characteristic wavelength at which a visual pigment absorbs light is regulated by interactions between protein (opsin) and retinylidene Schiff base chromophore. By using site-directed mutagenesis, charged amino acids in bovine rhodopsin transmembrane helix C were systematically replaced. Substitution of glutamic acid-134 or arginine-135 did not affect spectral properties. However, substitution of glutamic acid-122 by glutamine or by aspartic acid formed pigments that were blue-shifted in light absorption (lambda max = 480 nm and 475 nm, respectively). While the substitution of glutamic acid-113 by aspartic acid gave a slightly red-shifted pigment (lambda max = 505 nm), replacement by glutamine formed a pigment that was strikingly blue-shifted in light absorption (lambda max = 380 nm). The 380-nm species existed in a pH-dependent equilibrium with a 490-nm species such that at acidic pH all of the pigment was converted to lambda max = 490 nm. We conclude that glutamic acid-113 serves as the retinylidene Schiff base counterion in rhodopsin. We believe that this opsin-chromophore interaction is an example of a general mechanism of color regulation in the visual pigments.