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Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.

Cysteines were introduced, one at a time, at amino acid positions 55-75 in the cytoplasmic region connecting helices I and II in rhodopsin. In each of the 21 cysteine mutants, the reactive native cysteine residues (C140 and C316) were replaced by serine. Except for N55C, all mutants formed rhodopsin-like chromophores and had normal photobleaching characteristics. The efficiency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The reactivity of the substituted cysteine in each mutant toward 4, 4'-dithiodipyridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and I75C were unreactive to 4-PDS under the conditions used, suggesting that they are embedded in the micelle or protein interior. The mutants V63C, H65C-T70C, and N73C reacted rapidly, while the remainder of the mutants reacted more slowly, and varied in reactivity with sequence position. For the mutants derivatized with 4-PDS, the rate of release of thiopyridone from the resulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investigated in the dark and in the light. Marked changes in the rates of thiopyridone release in the light were found at specific sites. Collectively, the data reveal tertiary interactions of the residues in the sequence investigated and demonstrate structural changes due to photoactivation.

The positions of single amino acids in the interhelical loop regions and the C-terminal tail of bacteriorhodopsin (bR) were investigated by X-ray diffraction using site-directed heavy-atom labeling. Since wild-type bR does not contain any cysteines, appropriate cysteine mutants were produced with a unique sulfhydryl group at specific positions. These sites were then labeled with mercury using the sulfhydryl specific reagent p-chloromercuribenzoate (p-CMB). The cysteine mutants D96A/V101C, V130C, A160C, and G231C were derivatized with labeling stoichiometries of 0.93 +/- 5%, 0.85 +/- 5%, 0.79 +/- 7%, and 0.77 +/- 8%, respectively (Hg per bR). No incorporation was observed with wild-type bR under the same conditions. All mutants and heavy-atom derivatives were fully active as judged by the kinetics of the photocycle and of the proton release and uptake. Moreover, the unit cell dimensions of the two-dimensional P3 lattice were unchanged by the mutations and the derivatization. This allowed the position of the mercury atoms, projected onto the plane of the membrane, to be calculated from the intensity differences in the X-ray diffraction pattern between labeled and unlabeled samples using Fourier difference methods. The X-ray diffraction data were collected at room temperature from oriented purple membrane films at 100% relative humidity without the use of dehydrating solvents. These native conditions of temperature, humidity, and solvent are expected to preserve the structure of the surface-exposed loops. Sharp maxima corresponding to a single mercury atom were found in the difference density maps for D96A/V101C and V130C. Residues 101 and 130 are in the short loops connecting helices C/D and D/E, respectively. No localized difference density was found for A160C and G231C. Residue 160 is in the longer loop connecting helices E and F, whereas residue 231 is in the C-terminal tail. Residues 160 and 231 are apparently in a more disordered and mobile part of the structure.

Previous mutagenesis studies have indicated the requirement of a tertiary structure in the intradiscal region with a disulfide bond between Cys-110 and Cys-187 for the correct assembly and/or function of rhodopsin. We have now studied a rhodopsin mutant in which only the natural intradiscal cysteines at positions 110, 185, and 187 are present while all the remaining seven cysteines in the wild-type bovine rhodopsin have been replaced by serines. The proteins formed on expression of this mutant in COS-1 cells bind 11-cis-retinal only partially to form the rhodopsin chromophore. We show that this is due to the presence of both correctly folded chromophore-forming opsin and misfolded opsins. Methods have been devised for the separation of the correctly folded and misfolded forms by selective elution from immunoaffinity adsorbants. Using several criteria, which include SDS-PAGE as well as UV/visible and CD spectroscopy, we find that the correctly folded mutant protein is indistinguishable in its spectral properties from the wild-type rhodopsin. Further, reaction of sulfhydryl groups in the correctly folded mutant pigment with N-ethylmaleimide indicates that alkylation of a single sulfhydryl requires denaturation or illumination, while reaction with an additional two sulfhydryl groups occurs only after reduction. The misfolded mutant opsins are characterized by reduced alpha-helical content, sulfhydryl reactivity under native conditions in the dark, and also the presence of a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of the light-induced proton release in bacteriorhodopsin/lipid/detergent micelles was monitored with the optical pH-indicator fluorescein bound covalently to positions 127-134 (helices D and E and the DE loop) on the extracellular side of the protein (the proton release side). Single cysteine residues were introduced in these positions by site-directed mutagenesis, and fluorescein was attached to the sulfhydryl group by reaction with (iodoacetamido)fluorescein. Two characteristic proton release times (approximately 20 and 70 microseconds) were observed. The faster time constant was recorded when fluorescein was attached to positions 127, 130, 131, 132, and 134, while the slower time was observed with the indicator bound to positions 128, 129, and 133. The results are rationalized by assuming specific helical wheel orientations for helics D and E and by making a choice for the residues in the DE loop: (i) The fast time constants occur with fluorescein either attached to residues 130, 131, and 132 that form the DE loop or when pointing toward the interior of the protein with its aqueous proton channel [residues 127 (helix D) and 134 (helix E)]. (ii) The slower time constants are detected with fluorescein exposed to the exterior lipid/detergent phase when bound to residues 128, 129 (both helix D), and 133 (helix E). This interpretation is supported by measurements of the polarity of the label environment which indicate for fluorescein in group i a more hydrophilic environment and for group ii a more hydrophobic environment. The fastest proton release time (10 microseconds) was observed with fluorescein bound to position 127.(ABSTRACT TRUNCATED AT 250 WORDS)

We present a method that allows the detection of the surface charge density of bacteriorhodopsin (bR) at any selected protein surface site. The optical pH indicator fluorescein was covalently bound to the sulfhydryl groups of single cysteine residues, which were introduced at selected positions in bR by site-directed mutagenesis. On the extracellular side, the positions were in the BC loop (72) and in the DE loop (129-134). On the cytoplasmic side, one position in each loop was labeled: 35 (AB), 101 (CD), 160 (EF), and 231 (carboxy tail). The apparent pKs of fluorescein in these positions were determined for various salt concentrations. The local surface charge density was calculated from the dependence of the apparent pK of the dye on the ionic strength using the Gouy-Chapman equation. The surface charge density at pH 6.6 is more negative on the cytoplasmic side (averaged over all positions, -2.5 +/- 0.2 elementary charges per bR) than on the extracellular side (average, -1.8 +/- 0.2 elementary charges per bR) with little variation along the surface. Since the experiments were performed with electrically neutral CHAPS/DMPC micelles, these values represent the charge present on bR itself.(ABSTRACT TRUNCATED AT 250 WORDS)

FTIR-difference spectroscopy in combination with site-directed mutagenesis has been used to investigate the role of water during the photocycle of bacteriorhodopsin. At least one water molecule is detected which undergoes an increase in H-bonding during the primary bR-->K phototransition. Bands due to water appear in the OH stretch region of the bR-->K FTIR-difference spectrum which downshift by approximately 12 cm-1 when the sample is hydrated with H2(18)O. In contrast to 2H2O, the H2(18)O-induced shift is not complete, even after 24 h of hydration. This indicates that even though water is still able to exchange protons with the outside medium, it is partially trapped in the interior of the protein. In the mutant Y57D, these bands are absent while a new set of bands appear at much lower frequencies which undergo H2(18)O-induced shifts. It is concluded that the water molecule we detect is located inside the bR active-site and may interact with Tyr-57. The change in its hydrogen-bonding strength is most likely due to the photoinduced all-trans-->13-cis isomerization of the retinal chromophore and the associated movement of the positively charged Schiff base during the bR-->K transition. In contrast, a second water molecule, whose infrared difference bands are not affected by the Y57D mutation, appears to undergo a decrease in hydrogen bonding during the K-->L and L-->M transitions.

Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.

The light-dark adaptation, photocycle kinetics, and acid-induced blue formation of the bacteriorhodopsin (bR) mutant Tyr-185-->Phe (Y185F) expressed in Halobacterium halobium have been investigated by both static and time-resolved visible absorption spectroscopy. Evidence is presented that a pH-dependent equilibrium exists between a bR570-like form (bRY185F570) and a red-shifted species in the light-adapted form of Y185F. In two related papers, we show that this species has vibrational features similar to the O intermediate. Key findings are that light adaptation causes formation of a purple species similar to bR570 and a second long-lived red-shifted species with a lambda max near 630 nm, well above the pH for the acid-induced blue transition. The concentration of the red-shifted species is pH- and salt-dependent, decreasing reversibly at high pH and high ionic strength. The dark-adapted state of Y185F also contains a small amount of the red-shifted species which is reversibly titratable. Dark adaptation is much slower than wild-type bR and causes a parallel decay of light-adapted bR and the red-shifted species. Time-resolved visible absorption spectroscopy reveals that the purple and the red-shifted species undergo separate photocycles. The purple species exhibits a relatively normal photocycle except for an increased rate of M formation kinetics. The red-shifted species has a photocycle involving a red-shifted K intermediate and a second longer lived intermediate possibly similar to N. The apparent absence of an O intermediate in the late photocycle of Y185F is attributed to cancellation by depletion bands due to the photoreacting red-shifted species.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytoplasmic loop connecting helices C and D in rhodopsin is a part of the region involved in protein-protein interactions during signal transduction. To probe the structure of the CD loop, we have replaced, one at a time, the amino acids 136-150 by cysteine residues. The cysteine substitution mutants contained only the introduced single reactive cysteines and were prepared from a base opsin mutant that retained only the three intradiscal cysteines. All of the cysteine substitution mutants formed the characteristic rhodopsin chromophore (lambda max, 500 nm) with 11-cis-retinal. They showed normal photobleaching characteristics and activated transducin in a light-dependent manner, albeit at lower levels than the wild-type pigment. The newly introduced cysteines in the substitution mutants all underwent alkylation in the dark with the membrane-permeant sulfhydryl reagent N-ethylmaleimide, but with varying rates. The cysteine substitution mutants also showed prominent differences in alkylation with membrane-impermeant N-polymethylenecarboxylmaleimides of various alkyl chain lengths. Notably, derivatization of the cysteines in the mutants was not observed with the polar sulfhydryl reagents iodoacetic acid or iodoacetamide. These findings highlight intrinsic differences in both the reactivity and accessibility of the different cysteine residues in the CD loop and support the important role for a structure in the second cytoplasmic region of rhodopsin.

Autosomal dominant retinitis pigmentosa (ADRP) is a hereditary form of retinitis pigmentosa which accounts for about 15% of all types of the latter disease. Recently, close to 50 mutations, mostly point mutations, have been identified in the rhodopsin gene in ADRP patients. We have introduced these mutations in the synthetic bovine rhodopsin gene and herein report on the expression of the mutant genes in COS-1 cells and studies in vitro of the properties of the expressed opsins. The mutant phenotypes fall into three classes: Class I mutants are expressed in COS-1 cells at wild-type levels, form the normal rhodopsin chromophore with 11-cis-retinal, and are transported to the cell surface. However, on illumination, they activate transducin inefficiently. Class II mutants remain in the endoplasmic reticulum and do not bind 11-cis-retinal to form the chromophore. Class III mutants are expressed at low levels and form rhodopsin chromophore only poorly. They also remain in the endoplasmic reticulum and, as expected, show high mannose glycosylation. Nearly all of the mutants studied show abnormal sensitivity to light compared to the wild type, and they activate transducin less efficiently. We conclude that the majority of the ADRP mutants have folding defects.

We have used heavy-atom labeling and X-ray diffraction to localize a single amino acid in the integral membrane protein bacteriorhodopsin (bR). To provide a labeling site, we used the bR mutant, A103C, which contains a unique cysteine residue in the short loop between transmembrane alpha-helices C and D. The mutant protein was expressed in and purified from Halobacterium halobium, where it forms a two-dimensional crystalline lattice. In the lattice form, the protein reacted with the sulfhydryl-specific reagent p-chloromercuribenzoate (p-CMB) in a 1:0.9 stoichiometry to yield the p-mercuribenzoate derivative (A103C-MB). The functional properties of A103C and A103C-MB, including the visible absorption spectrum, light-dark adaptation, photocycle, and proton release kinetics, were similar to those of wild-type bR. X-ray diffraction experiments demonstrated that A103C and A103C-MB membranes have the same hexagonal protein lattice as wild-type purple membrane. Thus, neither the cysteine substitution nor mercury labeling detectably affected bR structure or function. By using Fourier difference methods, the in-plane position of the mercuribenzoate label was calculated from intensity differences in the X-ray diffraction patterns of A103C and A103C-MB. This analysis revealed a well-defined mercury peak located between alpha-helices C and D. The approach reported here offers promise for refining the bR structural model, for monitoring conformational changes in bR photointermediates, and for studying the structure of other proteins in two-dimensional crystals.

Butyldiphenylchlorosilanes react with deoxy- and N-protected deoxymononucleotides to give, in each case, the 3'-O-butyldiphenylsilyl ether in high yield. A comparative study of n-, sec-, and tert-butyldiphenylchlorosilanes showed, as expected, a wide variation in the rate of formation and in the stability of the 3'-O-silyl ethers, the tert-butylphenylsilyl group being the one of choice in polynucleotide synthesis. The silyl group can be readily removed on treatment with fluoride ion in buffered pyridine at room temperature. This property markedly increases flexibility of the current methodology for polynucleotide synthesis. Secondly, because of its high lipophilicity, the silyl group allows rapid solvent extraction of synthetic intermediates, and thirdly, it greatly facilitates the isolation of the required product in synthetic reactions because of the selective and strong retention of the condensation product during reverse-phase high-pressure liquid chromatography. Several examples of synthetic procedures, including a stepwise synthesis of an undecanucleotide, are given which demonstrate overall simplification of polynucleotide synthesis.

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.

Reverse phase high-pressure liquid chromatography (HPLC) using columns containing microparticulate materials with bonded octadecyl groups has been developed as a rapid and efficient method for the separation of nucleosides, nucleotides, and, in particular, of protected oligonucleotides which are standard intermediates in the stepwise synthesis of deoxyribopolynucleotides. Reported are extensive studies of the influence of the different purine and pyrimidine bases, of protecting groups, of the phosphate groups, and of the chain lengths of oligonucleotides on their retention on such columns. Further, the application of HPLC in the stepwise synthesis of an oligonucleotide, d(G-G-A-A-G-C-T-T-A-A-C), has been described. The methods, which are herein described, lend themselves to separations on a preparative scale and effect a marked reduction (up to 50%) in the time required for the synthesis of oligonucleotides.

Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.

In developing new approaches to structural studies of polytopic transmembrane proteins, we have prepared bacteriorhodopsin mutants containing single cysteine residues at selected sites in different topological domains. Four such mutants were prepared: Gly-72----Cys and Ser-169----Cys in the presumed looped-out regions on the opposite sides of the membrane bilayer and Thr-90----Cys and Leu-92----Cys in the membrane-embedded helix C. The four mutants folded and regenerated the characteristic chromophore in detergent/phospholipid micelles and pumped protons like the wild-type bacteriorhodopsin. After reconstitution in asolectin vesicles, the sulfhydryl groups in the mutants Gly-72----Cys and Ser-169----Cys reacted with iodo[2-3H]acetic acid, while the sulfhydryl groups in the membrane-embedded mutants, Thr-90----Cys and Leu-92----Cys, did not. The sulfhydryl groups in all four mutants could be derivatized in the denatured state by reaction with iodoacetic acid or 6-acryloyl-2-(dimethylamino)naphthalene. Of these derivatives, the two from the mutants Gly-72----Cys and Ser-169----Cys folded like the wild-type bacterioopsin, whereas of the two from the helix C mutants, Thr-90----Cys and Leu-92----Cys, only the latter folded normally. However, the folding of Leu-92----Cys was also impaired when treated with the bulky 5-(iodoacetamido)fluorescein. The reactivity and the folding behavior of the cysteine mutants can thus report on the topographic domain as well as on the orientation of the helices within the membrane.

Butyldiphenylchlorosilanes react with deoxy- and N-protected deoxymononucleotides to give, in each case, the 3'-O-butyldiphenylsilyl ether in high yield. A comparative study of n-, sec-, and tert-butyldiphenylchlorosilanes showed, as expected, a wide variation in the rate of formation and in the stability of the 3'-O-silyl ethers, the tert-butylphenylsilyl group being the one of choice in polynucleotide synthesis. The silyl group can be readily removed on treatment with fluoride ion in buffered pyridine at room temperature. This property markedly increases flexibility of the current methodology for polynucleotide synthesis. Secondly, because of its high lipophilicity, the silyl group allows rapid solvent extraction of synthetic intermediates, and thirdly, it greatly facilitates the isolation of the required product in synthetic reactions because of the selective and strong retention of the condensation product during reverse-phase high-pressure liquid chromatography. Several examples of synthetic procedures, including a stepwise synthesis of an undecanucleotide, are given which demonstrate overall simplification of polynucleotide synthesis.

Reverse phase high-pressure liquid chromatography (HPLC) using columns containing microparticulate materials with bonded octadecyl groups has been developed as a rapid and efficient method for the separation of nucleosides, nucleotides, and, in particular, of protected oligonucleotides which are standard intermediates in the stepwise synthesis of deoxyribopolynucleotides. Reported are extensive studies of the influence of the different purine and pyrimidine bases, of protecting groups, of the phosphate groups, and of the chain lengths of oligonucleotides on their retention on such columns. Further, the application of HPLC in the stepwise synthesis of an oligonucleotide, d(G-G-A-A-G-C-T-T-A-A-C), has been described. The methods, which are herein described, lend themselves to separations on a preparative scale and effect a marked reduction (up to 50%) in the time required for the synthesis of oligonucleotides.

Millisecond photocycle kinetics were measured at room temperature for 13 site-specific bacteriorhodopsin mutants in which single aspartic acid residues were replaced by asparagine, glutamic acid, or alanine. Replacement of aspartic acid residues expected to be within the membrane-embedded region of the protein (Asp-85, -96, -115, or -212) produced large alterations in the photocycle. Substitution of Asp-85 or Asp-212 by Asn altered or blocked formation of the M410 photointermediate. Substitution of these two residues by Glu decreased the amount of M410 formed. Substitutions of Asp-96 slowed the decay rate of the M410 photointermediate, and substitutions of Asp-115 slowed the decay rate of the O640 photointermediate. Corresponding substitutions of aspartic acid residues expected to be in cytoplasmic loop regions of the protein (Asp-36, -38, -102, or -104) resulted in little or no alteration of the photocycle. Our results indicate that the defects in proton pumping which we have previously observed upon substitution of Asp-85, Asp-96, Asp-115, and Asp-212 [Mogi, T., Stern, L. J., Marti, T., Chao, B. H., & Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4148-4152] are closely coupled to alterations in the photocycle. The photocycle alterations observed in these mutants are discussed in relation to the functional roles of specific aspartic acid residues at different stages of the bacteriorhodopsin photocycle and the proton pumping mechanism.