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M S Braiman

Publications and source records attributed to M S Braiman.

At least 19 recordsLinked to original sources
PubMed1998

Arginine to glutamine substitutions in the fourth module of Xenopus interphotoreceptor retinoid-binding protein.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) is unusual for a lipid-binding protein in that its gene is expressed uniquely by cells of photoreceptor origin and consists of four homologous repeats, each coding for a module of approximately 300 amino acid residues. All-trans retinol binding domains, which appear to be present in each module, are composed of conserved hydrophobic regions [Baer et al, Exp Eye Res 1998; 66:249-262]. Here we investigate the role of highly conserved arginines contained in these regions. METHODS: To study the arginines in an individual module without the interference of ligand-binding activity elsewhere in the protein, we expressed in E. coli the fourth module of Xenopus IRBP by itself as a soluble thioredoxin fusion protein (X4IRBP). Arginines 1005, 1041, 1073 and 1122 were separately replaced by glutamine using PCR overlap extension mutagenesis. The glutamine substitutions were confirmed by liquid chromatography-tandem mass spectrometry. The binding of all-trans retinol and 9-(9-anthroyloxy)stearic acid (9-AS) to X4IRBP and each of the mutants was evaluated by fluorescence spectroscopy. Binding was followed by monitoring the enhancement of ligand fluorescence and the quenching of protein endogenous fluorescence. The ability of the recombinant proteins to protect all-trans retinol from oxidative degradation was evaluated by monitoring absorbance at 325 nm over time. RESULTS: The substitution of Gln for Arg1005 about doubled the amount of ligand necessary to attain saturation and about doubled the level of fluorescence enhancement obtained at saturation for both all-trans retinol and 9-AS. Although there was not a significant change in the Kd, the substitution increased the calculated number of binding sites (N) from approximately 2 to approximately 4 per polypeptide. The other Arg->Gln mutants did not significantly change the Kd or N. None of the mutations compromised the ability of the module to protect all-trans retinol from degradation. CONCLUSIONS: Our data suggest that the function of the conserved arginines in IRBP is fundamentally different from that of other retinoid-binding proteins. These residues do not appear to play a role in defining the specificity of the ligand-binding domain. Rather, Arg1005 appears to play a role in defining the capacity of the domain. Our data suggest that the binding site consists of a single hydrophobic cavity promiscuous for fatty acids and all-trans retinol.

Amino Acid Substitution↗
PubMed1998

Soluble expression in E. coli of a functional interphotoreceptor retinoid-binding protein module fused to thioredoxin: correlation of vitamin A binding regions with conserved domains of C-terminal processing proteases.

The exchange of all-trans retinol and 11-cis retinal between the photoreceptors and retinal pigmented epithelium is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP contains binding sites for retinoids, docosahexaenoic acid and probably cell surface and matrix receptors. IRBP arose through the quadruplication of an ancient protein, represented by its carboxy-terminal module (module 4 in amphibians and mammals). Module 4 has retinol binding activity and is composed of regions coded for by each of IRBP's four exons. Determining the function of the exons has been hampered by insoluble expression of module 4 in Escherichia coli. Here, we found that module 4 of Xenopus IRBP (X4IRBP), as well as its exon segments, can be expressed in a soluble form as thioredoxin fusion proteins. The recombinant proteins were purified by ion exchange and arsenical-based affinity chromatography. Liquid chromatography/mass spectrometry confirmed that the sequence of X4IRBP is correct. All-trans retinol binding was characterized by monitoring enhancement of retinol fluorescence, quenching of intrinsic protein fluorescence, and transfer of energy to the bound retinol. Retinol bound to X4IRBP at 2.20+/-0.29 sites with a KD=1.25+/-0.39. One of the two sites was localized to Exons(2+3) and had a KD=0.26+/-0.13 micron. This site, which supported protein quenching and energy transfer, probably contains at least one of the two conserved tryptophans present in this segment. The second site was localized to Exon 4. This site supported the enhancement of retinol fluorescence but not protein quenching or energy transfer and had a KD=1.94+/-0.20 micron. Exon 1 had no retinol binding activity. The location of the retinol binding regions correlated with the distribution of domains conserved between IRBPs and the newly recognized family of C-terminal processing proteases (CtpAs), proteins which bind and cleave non-polar carboxy termini.

Amino Acid Sequence↗
PubMed1998

Fourth module of Xenopus interphotoreceptor retinoid-binding protein: activity in retinoid transfer between the retinal pigment epithelium and rod photoreceptors.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), an extracellular protein believed to support the exchange of retinoids between the neural retina and retinal pigment epithelium (RPE) in the vertebrate eye, exhibits a modular, i.e., repeat, structure. The present study was undertaken to determine whether an individual module of IRBP has activity in retinoid transfer between the RPE and rod photoreceptors. METHODS: The retinoid transfer activity of a recombinant protein corresponding to the fourth module of Xenopus laevis IRBP (X4IRBP) was examined in two ways. First, X4IRBP was tested for its ability to support the regeneration of porphyropsin in detached/reattached Xenopus retina/RPE-eyecups. Following illumination and removal of native IRBP, Xenopus eyecups supplemented with 42 microM X4IRBP or (as a control) Ringer's solution were incubated in darkness and then analyzed for regenerated porphyropsin. Second, toad (Bufo marinus) RPE-eyecup preparations were used to evaluate X4IRBP's ability to promote the release of 11-cis retinal from the RPE. RESULTS: The regeneration of porphyropsin in X4IRBP-supplemented Xenopus retina/RPE-eyecups (0.45 +/- 0.04 nmol; mean +/- SEM, n = 11) exceeded that in controls (0.13 +/- 0.02 nmol, n = 11). For promoting the release of 11-cis retinal from the toad RPE, 42 microM X4IRBP was more effective than equimolar bovine serum albumin although considerably less than that of 26 microM native bovine IRBP. CONCLUSIONS: The results indicate a low but significant activity of IRBP's fourth module in reactions relevant to retinoid exchange.

Animals↗
PubMed1997

Vibrational spectra of individual millimeter-size membrane patches using miniature infrared waveguides.

We have used miniature planar IR waveguides, consisting of Ge strips 30-50 microm thick and 2 mm wide, as evanescent-wave sensors to detect the mid-(IR) evanescent-wave absorbance spectra of small areas of biomolecular monolayers and multilayers. Examples include picomolar quantities of an integral transmembrane protein (bacteriorhodopsin) and lipid (dimyristoyl phosphatidylcholine). IR bands due to the protein and lipid components of the plasma membrane of individual 1.5-mm-diameter devitellinized Xenopus laevis oocytes, submerged in buffer and sticking to the waveguide surface, were also detected. A significant improvement in sensitivity was observed, as compared to previous sizes and geometries of evanescent-wave sensors (e.g., commercially available internal reflection elements or tapered optical fibers). These measurements suggest the feasibility of using such miniature supported planar IR waveguides to observe structural changes in transmembrane proteins functioning in vivo in single cells.

Animals↗
PubMed1997

Nano- and microsecond time-resolved FTIR spectroscopy of the halorhodopsin photocycle.

Step-scan Fourier transform infrared spectroscopy with 50 ns time resolution was applied to the early stages of the photocycle of halorhodopsin (hR) for the temperature range 3-42 degrees C. Kinetic data analysis with global fitting revealed two distinct kinetic processes associated with relaxations of the early red-shifted photoproduct hK; these processes have time constants tau 1 approximately equal to 280 ns and tau 2 approximately equal to 360 microns at 20 degrees C. Spectral features demonstrate that the tau 1 process corresponds to a transition between two distinct bathointermediates, hKE and hKL. The vibrational difference bands associated with both tau 1 and tau 2 transitions are spread throughout the whole 1800-900 cm-1 range. However, the largest bands correspond to ethylenic C=C stretches, fingerprint C-C stretches and hydrogen out-of-plane (HOOP) wags of the retinal chromophore. The time evolution of these difference bands indicate that both the tau 1 and tau 2 decay processes involve principally a relaxation of the chromophore and its immediate environment. The decay of the intense HOOP vibrations is nearly equally divided between the tau 1 and tau 2 processes, indicating a complex chromophore relaxation from a twisted nonrelaxed conformation in the primary (hKE) bathointermediate, to a less-twisted structure in hKL, and finally to a roughly planar structure in the hypsochromically shifted hL intermediate. This conclusion is also supported by the unexpectedly large positive entropy of activation observed for the tau 1 process. The two relaxations from hKE to hL are largely analogous to corresponding relaxations (KE-->KL-->L) in the bacteriorhodopsin photocycle, except that the second step is slowed down by over 200-fold in hR.

Bacteriorhodopsins↗
PubMed1996

A large photolysis-induced pKa increase of the chromophore counterion in bacteriorhodopsin: implications for ion transport mechanisms of retinal proteins.

The proton-pumping mechanism of bacteriorhodopsin is dependent on a photolysis-induced transfer of a proton from the retinylidene Schiff base chromophore to the aspartate-85 counterion. Up until now, this transfer was ascribed to a > 7-unit decrease in the pKa of the protonated Schiff base caused by photoisomerization of the retinal. However, a comparably large increase in the pKa of the Asp-85 acceptor also plays a role, as we show here with infrared measurements. Furthermore, the shifted vibrational frequency of the Asp-85 COOH group indicates a transient drop in the effective dielectric constant around Asp-85 to approximately 2 in the M photointermediate. This dielectric decrease would cause a > 40 kJ-mol-1 increase in free energy of the anionic form of Asp-85, fully explaining the observed pK alpha increase. An analogous photolysis-induced destabilization of the Schiff base counterion could initiate anion transport in the related protein, halorhodopsin, in which aspartate-85 is replaced by Cl- and the Schiff base proton is consequently never transferred.

Aspartic Acid↗
PubMed1994

Infrared spectroscopic detection of light-induced change in chloride-arginine interaction in halorhodopsin.

A light-induced transient change in the ionic interaction between chloride and arginine in the transmembrane anion pump halorhodopsin (hR) is detected with infrared absorption spectroscopy. In the IR difference spectrum of hR and one of its photoproducts (hL), only a few bands have frequencies that depend on the particular halide ion (Cl-, Br-, or I-) present. Three of the halide-sensitive negative difference bands (at 1695, 1610, and 1170 cm-1) correspond in frequency to arginine C-N vibrations and undergo anion-dependent shifts that match those seen in ethylguanidinium halide model compounds. These shifts reflect the different strengths of the ionic interactions formed with the various halides. We conclude that a halide-arginine ion pair is present in the hR state; this interaction appears to be disrupted by photoconversion to hL.

Anions↗
PubMed1994

Anion-protein interactions during halorhodopsin pumping: halide binding at the protonated Schiff base.

Halorhodopsin (hR), the light-driven chloride pump of Halobacterium halobium, has been studied by Fourier transform infrared (FTIR) spectroscopy. Direct hydrogen bonding of halide ions with the protonated Schiff base (PSB) group was detected by means of halide-dependent perturbations on this group's vibrational frequencies. FTIR difference spectra were obtained of the hR-->hL photoreaction in reconstituted membrane vesicles. Nearly identical results were obtained using either low-temperature static difference spectroscopy at 1-cm-1 resolution or a stroboscopic time-resolved technique with 5-ms temporal and 2-cm-1 spectral resolution. The frequency of the negative difference band due to the PSB C = N stretch mode in the hR state shows a dependence on the type of halide counteranion that is present, 1632 cm-1 in the presence of Cl-, 1631 cm-1 in Br-, and 1629 cm-1 in I-. The C = NH+ stretch frequency thus correlates with the strength of the hydrogen bond formed by the halide. Analogous halide-dependent shifts of the C = NH+ frequency were observed in IR spectra of model compound retinylidene PSB salts. We also observed a significant halide dependence of the visible absorption maximum of hR solubilized in lauryl maltoside detergent. From such halide perturbation effects, we conclude that there is a direct hydrogen-bonded interaction between the Schiff base group and an externally supplied halide ion in the hR state. Halide perturbation effects are also observed for PSB-group vibrations in the hL state. Thus, despite an apparent overall weakening of hydrogen-bonding interactions of the PSB with its environment after chromophore photoisomerization to form hL, the PSB remains hydrogen-bonded to the halide. The results are best explained in terms of a "one-site, two-state" model for anion binding near the chromophore in the hR state, as opposed to a previously proposed two-site model.

Anions↗
PubMed1994

Expression and characterization of the fourth repeat of Xenopus interphotoreceptor retinoid-binding protein in E. coli.

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRBP's 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 microM). The recombinant fourth repeat of Xenopus IRBP binds [3H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a Kd = 0.4 microM. The expression of recombinant IRBP fragments as fusion proteins in prokaryotes will be useful for defining the structural requirements for ligand binding by this interesting protein.

Animals↗
PubMed1993

Proton transfer from Asp-96 to the bacteriorhodopsin Schiff base is caused by a decrease of the pKa of Asp-96 which follows a protein backbone conformational change.

In the bacteriorhodopsin photocycle the transported proton crosses the major part of the hydrophobic barrier during the M to N reaction; in this step the Schiff base near the middle of the protein is reprotonated from D96 located near the cytoplasmic surface. In the recombinant D212N protein at pH > 6, the Schiff base remains protonated throughout the photocycle [Needleman, Chang, Ni, Váró, Fornés, White, & Lanyi (1991) J. Biol. Chem. 266, 11478-11484]. Time-resolved difference spectra in the visible and infrared are described by the kinetic scheme BR-->K<==>L<==>N (-->N')-->BR. As evidenced by the large negative 1742-cm-1 band of the COOH group of the carboxylic acid, deprotonation of D96 in the N state takes place in spite of the absence of the unprotonated Schiff base acceptor group of the M intermediate. Instead of internal proton transfer to the Schiff base, the proton is released to the bulk, and can be detected with the indicator dye pyranine during the accumulation of N'. The D212N/D96N protein has a similar photocycle, but no proton is released. As in wild-type, deprotonation of D96 in the N state is accompanied by a protein backbone conformational change indicated by characteristic amide I and II bands. In D212N the residue D96 can thus deprotonate independent of the Schiff base, but perhaps dependent on the detected protein conformational change. This could occur through increased charge interaction between D96 and R227 and/or increased hydration near D96. We suggest that the proton transfer from D96 to the Schiff base in the wild-type photocycle is driven also by such a decrease in the pKa of D96.

Amino Acid Sequence↗
PubMed1992

Structural comparison of metarhodopsin II, metarhodopsin III, and opsin based on kinetic analysis of Fourier transform infrared difference spectra.

Fourier transform infrared difference spectra were measured at 30-s intervals after a complete bleach of rhodopsin (rho) samples at 20 degrees C and three different pH values. At each pH, all of the spectra could be fit globally to two exponential decay processes. Using a branched unimolecular kinetic model in which metarhodopsin II (meta II) is hydrolyzed to opsin and retinal both directly and through metarhodopsin III (meta III), we calculated rho-->meta II, rho-->meta III, and rho-->opsin difference spectra at each of the pH values and obtained estimates for the microscopic rate constants at each pH. Because of assumptions that had to be made about the branching ratio between the meta II decay pathways, some uncertainties remain in our calculated rho-->meta III difference spectrum at each pH. Nevertheless, our data covering long time ranges, especially those obtained at pH 8, place significant new constraints on the spectrum of meta III and thus on its structure. The rho-->meta II spectrum shows no significant pH dependence over the range examined (pH 5.5-8). However, the rho-->meta III and rho-->opsin spectra each include a limited subset of pH-dependent peaks, which are mostly attributable to titratable amino acid side chains. Our observations can be used to refine an earlier conclusion that the visual pigment refolds to a rhodopsin-like conformation during meta II decay (Rothschild, K.J., J. Gillespie, and W.J. DeGrip. 1987 Biophys. J. 51:345-350). Most of this refolding occurs in the same way at pH values ranging from 5.5 to 8 and whether meta II decays to meta III or opsin. Meta II displays unique spectral perturbations that are mostly attributable to a few residues, probably including three to four aspartic or glutamic acids and an arginine.

Animals↗
PubMed1991

Protein dynamics in the bacteriorhodopsin photocycle: submillisecond Fourier transform infrared spectra of the L, M, and N photointermediates.

The usefulness of stroboscopic time-resolved Fourier transform IR spectroscopy for studying the dynamics of biological systems is demonstrated. By using this technique, we have obtained broadband IR absorbance difference spectra after photolysis of bacteriorhodospin with a time resolution of approximately 50 microseconds, spectral resolution of 4 cm-1, and a detection limit of delta A less than or equal to 10(-4). These capabilities permit observation of detailed structural changes in individual residues as bacteriorhodopsin passes through its L, M, and N intermediate states near physiological temperatures. When combined with band assignments based on isotope labeling and site-directed mutagenesis, the stroboscopic Fourier transform IR difference spectra show that on the time scale of the L intermediate, Asp-96 has an altered environment that may be accompanied by change in its protonation state. On the time scale of the L----M transition, this Asp-96 perturbation/deprotonation is largely reversed, and Asp-85 becomes protonated. During the M----N transition, Asp-85 appears to remain protonated but undergoes a change in its environment as evidenced by a shift of vC = O from 1761 to 1755 cm-1. The retention of a proton on Asp-85 in the N state indicates that the proton transferred from the Schiff base to this residue in the L----M step is not released to the extracellular medium during the same photocycle, but rather during a subsequent one. Also during the M----N transition, Asp-96 undergoes a deprotonation (possibly for the second time in a single photocycle). Bands in the amide I and amide II spectral regions in the M----N difference spectrum indicate the occurrence of a conformational change involving one or more peptide groups in the protein backbone.

Bacteriorhodopsins↗
PubMed1990

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism.

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.

Amino Acid Sequence↗
PubMed1989

Vibrational spectroscopy of bacteriorhodopsin mutants: chromophore isomerization perturbs tryptophan-86.

Fourier transform infrared difference spectra have been obtained for the bR----K and bR----M photoreactions of bacteriorhodopsin mutants with Phe replacements for Trp residues 10, 12, 80, 86, 138, 182, and 189 and Cys replacements for Trp residues 137 and 138. None of the tryptophan mutations caused a significant shift in the retinylidene C = C or C-C stretching frequencies of the visible absorption maximum of the chromophore, it is concluded that none of the tryptophan residues are essential for forming a normal bR570 chromophore. However, a 742-cm-1 negative peak attributed previously to the perturbation of a tryptophan residue during the bR----K photoreaction was found to be absent in the bR----K and bR----M difference spectra of the Trp-86 mutant. On this basis, we conclude that the structure or environment of Trp-86 is altered during the bR----K photoreaction. All of the other Trp----Phe mutants exhibited this band, although its frequency was altered in the Trp-189----Phe mutant. In addition, the Trp-182----Phe mutant exhibited much reduced formation of normal photoproducts relative to the other mutants, as well as peaks indicative of the presence of additional chromophore conformations. A model of bR is discussed in which Trp-86, Trp-182, and Trp-189 form part of a retinal binding pocket. One likely function of these tryptophan groups is to provide the structural constraints needed to prevent chromophore photoisomerization other than at the C13 = C14 double bond.

Amino Acid Sequence↗
PubMed1989

Conserved amino acids in F-helix of bacteriorhodopsin form part of a retinal binding pocket.

A 3-dimensional model for the retinal binding pocket in the light-driven proton pump, bacteriorhodopsin, is proposed on the basis of spectroscopic studies of bacteriorhodopsin mutants. In this model Trp-182, Pro-186 and Trp-189 surround the polyene chain while Tyr-185 is positioned close to the retinylidene Schiff base. This model is supported by sequence homologies in the F-helices of bacteriorhodopsin and the related retinal proteins, halorhodopsin and rhodopsins.

Amino Acids↗
PubMed1988

Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212.

Fourier transform infrared (FTIR) difference spectra have been obtained for the bR----K, bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu. Difference peaks that had previously been attributed to Asp COOH groups on the basis of isotopic labeling were absent or shifted in these mutants. In general, each COOH peak was affected strongly by mutation at only one of the four residues. Thus, it was possible to assign each peak tentatively to a particular Asp. From these assignments, a model for the proton-pumping mechanism of bR is derived, which features proton transfers among Asp-85, -96, and -212, the chromophore Schiff base, and other ionizable groups within the protein. The model can explain the observed COOH peaks in the FTIR difference spectra of bR photointermediates and could also account for other recent results on site-directed mutants of bR.

Aspartic Acid↗
PubMed1988

Fourier transform infrared study of the halorhodopsin chloride pump.

Halorhodopsin (hR) is a light-driven chloride pump located in the cell membrane of Halobacterium halobium. Fourier transform infrared difference spectroscopy has been used to study structural alterations occurring during the hR photocycle. The frequencies of peaks attributed to the retinylidene chromophore are similar to those observed in the spectra of the related protein bacteriorhodopsin (bR), indicating that in hR as in bR an all-trans----13-cis isomerization occurs during formation of the early bathoproduct. Spectral features due to protein structural alterations are also similar for the bR and hR photocycles. For example, formation of the red-shifted primary photoproducts of both hR and bR results in similar carboxyl peaks in the 1730-1745-cm-1 region. However, in contrast to bR, no further changes are observed in the carboxyl region during subsequent steps in the hR photocycle, indicating that additional carboxyl groups are not directly involved in chloride translocation. Overall, the close similarity of vibrations in hR and bR photoproduct difference spectra supports the existence of some common elements in the molecular mechanisms of energy transduction and active transport by these two proteins.

Bacteriorhodopsins↗