Hydrogen exchange study of membrane-bound rhodopsin. II. Light-induced protein structure change.

Hydrogen exchange studies of rhodopsin in disc membranes demonstrated that photolysis induces changes in the protein itself. Two different altered forms were detected. A late photointermediate in the bleaching sequence, which can be identified with metarhodopsin II, displays accelerated exchange. Subsequently, at the stage of fully bleached opsin, exchange becomes even slower than in rhodopsin. These changes involve only a small fraction of the protein's internally hydrogen-bonded peptide groups. The unusually large fraction of exposed peptide hydrogens observed previously for rhodopsin is unaltered in the photolyzed forms.     

Re-examination of rhodopsin structure by hydrogen exchange

The hydrogen exchange behavior of rhodopsin was re-examined by studies of the protein in the disc membrane and after solubilization in octyl glucoside. The methods used measure either the peptide hydrogens alone (hydrogen-deuterium exchange by infrared spectroscopy) or all slowly exchanging hydrogens (hydrogen-tritium exchange by hel filtration). Under mild exchange conditions, disc membranes and solubilized lipid-free proteins show very similar exchange behavior, indicating the absence of slowly exchanging lipid protons. At high temperature, exchange of an additional large group of very slow peptide NH can be detected. The total number of slow hydrogens significantly exceeds the amide content, and apparently includes slowly exchanging protons from perhaps 40% of the protein's non-amide side chains. This is thought to require the involvement of many polar side chains in internal H-bonding. The exchange rates of the non-amide side chains sites have not been determined. However, to the extent that these contribute to the fast time region of the measured kinetic H-exchange curve, previously identified with exposed, non-H-bonded peptides, the estimate of freely exposed rhodopsin peptides must be reduced. The fraction of free peptides could range from a remarkably high value of 70% down to about 45%.     

Reactions of the sulfhydryl groups of membrane-bound bovine rhodopsin.

Reactions of the sulfhydryl groups of bovine rhodopsin in rod outer segment membranes have been investigated using 4,4'-dithiopyridine. This reagent is uncharged at neutral pH and rapidly equilibrates across phospholipid bilayers. Membrane-bound rhodopsin has two kinetically distinguishable sulfhydryl groups reactive to the reagent, this stoichiometry being unchanged by bleaching provided the sulfhydryl reactions themselves are carried out in the dark. The rates of the reactions, however, are substantially increased by bleaching. Irradiation of bleached membranes, either with white light or wavelengths in the neighborhood of 475 nm, results in an increase in the number of reactive sulfhydryls relative to that found for bleached membranes in the dark. A component of the light-driven reaction is dependent on the Ca2+ content of the medium.     

Primary structure of sensory rhodopsin I, a prokaryotic photoreceptor.

The gene coding for sensory rhodopsin I (SR-I) has been identified in a restriction fragment of genomic DNA from the Halobacterium halobium strain L33. Of the 1014 nucleotides whose sequence was determined, 720 belong to the structural gene of SR-I. In the 5' non-coding region two putative promoter elements and a ribosomal binding site have been identified. The 3' flanking region bears a potential terminator structure. The SR-I protein moiety carries no signal peptide and is not processed at its N terminus. The C terminus, however, lacks the last aspartic acid residue encoded by the gene. Analysis of the primary structure of SR-I reveals no consistent homology with the eukaryotic photoreceptor rhodopsin, but 14% homology with the halobacterial ion pumps, bacteriorhodopsin (BR) and halorhodopsin (HR). Residues conserved in all three proteins are discussed with respect to their contribution to secondary structure, retinal binding and ion translocation. The aspartic acid residue which mediates in BR the reprotonation of the Schiff base (D96) is replaced in SR-I by a tyrosine (Y87). This amino acid replacement is proposed to be of crucial importance in the evolution of the slow-cycling photosensing pigment SR-I.     

Immobilization of native membrane-bound rhodopsin on biosensor surfaces

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.     

Hydrogen ion changes of rhodopsin I. Proton uptake during the metarhodopsin I 478 metarhodopsin II 308 reaction

The hydrogen ion changes resulting from the photolysis of the rod visual pigment, rhodopsin, have been investigated. Low temperature was used to isolate the metarhodopsin I₄₇₈ to II₃₈₀ reaction of rhodopsin and indicator dye was used to simultaneously measure the hydrogen ion changes of the rhodopsin solution.The results indicate that illuminated rhodopsin takes up a proton during the metarhodopsin I₄₇₈ to II₃₈₀ reaction and releases protons at later intermediate stages. The results are consistent with data indicating pK changes of rhodopsin as the basis for the R₂ phase of the early receptor potential and hydrogen ion changes of the medium or pK changes of rhodopsin as having effects on the late receptor potential.     

Protein secondary structure and homology by neural networks. The alpha-helices in rhodopsin

Neural networks provide a basis for semiempirical studies of pattern matching between the primary and secondary structures of proteins. Networks of the perceptron class have been trained to classify the amino-acid residues into two categories for each of three types of secondary feature: alpha-helix or not, beta-sheet or not, and random coil or not. The explicit prediction for the helices in rhodopsin is compared with both electron microscopy results and those of the Chou-Fasman method. A new measure of homology between proteins is provided by the network approach, which thereby leads to quantification of the differences between the primary structures of proteins.     

Hydrogen exchange and the dynamic structure of proteins

Summary In native proteins, buried, labile protons undergo isotope exchange with solvent hydrogens, but the kinetics of exchange are markedly slower than in unfolded polypeptides. This indicates that, whereas buried protein atoms are shielded from solvent, the protein fluctuates around the time average structure and occasionally exposes buried sites to solvent. Generally, hydrogen exchange studies are designed to characterize the nature of the fluctuations between conformational substates, to monitor the shift in conformational equilibria among protein substates due to ligand binding or other factors, or to monitor the major cooperative denaturation transition. In this article, we review the recent reports of hydrogen exchange in proteins, focusing on recent advances in methodology, especially with regard to the implications of the results for the mechanism of hydrogen exchange in folded proteins.     

Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.     

Hydrogen exchange methods to study protein folding

The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed information so far available on the structure and properties of protein folding intermediates. Direct HX measurements can define the structure of tenuous molten globule forms that are generally inaccessible to the usual crystallographic and NMR methods (C. Redfield review in this issue). HX pulse labeling methods can specify the structure, stability and kinetics of folding intermediates that exist for less than 1 s during kinetic folding. Native state HX methods can detect and characterize folding intermediates that exist as infinitesimally populated high energy excited state forms under native conditions. The results obtained in these ways suggest principles that appear to explain the properties of partially folded intermediates and how they are organized into folding pathways. The application of these methods is detailed here.     

Light-stable rhodopsin. I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative.

With the aim of preparing a light-stable rhodopsin-like pigment, an analog, II, of 11-cis retinal was synthesized in which isomerization of the C11-C12 cis-double bond is blocked by a cyclohexene ring built around the C10 to C13-methyl. The analog II formed a rhodopsin-like pigment (rhodopsin-II) with opsin expressed in COS-1 cells and with opsin from rod outer segments. The rate of rhodopsin-II formation from II and opsin was approximately 10 times slower than that of rhodopsin from 11-cis retinal and opsin. After solubilization in dodecyl maltoside and immunoaffinity purification, rhodopsin-II displayed an absorbance ratio (A280nm/A512nm) of 1.6, virtually identical with that of rhodopsin. Acid denaturation of rhodopsin-II formed a chromophore with lambda max, 452 nm, characteristic of protonated retinyl Schiff base. The ground state properties of rhodopsin-II were similar to those of rhodopsin in extinction coefficient (41,200 M-1 cm-1) and opsin-shift (2600 cm-1). Rhodopsin-II was stable to hydroxylamine in the dark, while light-dependent bleaching by hydroxylamine was slowed by approximately 2 orders of magnitude relative to rhodopsin. Illumination of rhodopsin-II for 10 s caused approximately 3 nm blue-shift and 3% loss of visible absorbance. Prolonged illumination caused a maximal blue-shift up to approximately 20 nm and approximately 40% loss of visible absorbance. An apparent photochemical steady state was reached after 12 min of illumination. Subsequent acid denaturation indicated that the retinyl Schiff base linkage was intact. A red-shift (approximately 12 nm) in lambda max and a 45% recovery of visible absorbance was observed after returning the 12-min illuminated pigment to darkness. Rhodopsin-II showed marginal light-dependent transducin activation and phosphorylation by rhodopsin kinase.     

The structure of bovine rhodopsin

We have isolated 16 peptides from a cyanogen bromide digest of rhodopsin. These cyanogen bromide peptides account for the complete composition of the protein. Methionine-containing peptides from other chemical and enzymatic digests of rhodopsin have allowed us to place the cyanogen bromide peptides in order, yielding the sequence of the protein. We have completed the sequence of most of the cyanogen bromide peptides. This information, in conjunction with that from other laboratories, forms the basis for our prediction of the secondary structure of the protein and how it may be arranged in the disk membrane.     

Hydrogen exchange study of some polynucleotides and transfer RNA

The apparent disagreement between published transfer RNA hydrogen exchange results and the tRNA cloverleaf model, prompted a re-investigation of the relationship between hydrogen exchange data and nucleic acid structure. Hydrogen-tritium exchange experiments were carried out with samples of pure and mixed tRNA and with the synthetic polynucleotide bihelices: poly(rA) · poly(rU), poly(rI) · poly(rC), poly(rG) · poly(rC) and poly(dG) · poly (dC).     

Methyl-accepting protein associated with bacterial sensory rhodopsin I.

In vivo radiolabeling of Halobacterium halobium phototaxis mutants and revertants with L-[methyl-3H] methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in phototaxis. The lability of the radiolabeled bands to mild base treatment indicated that the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing phototaxis receptors in H. halobium. It was absent in a strain that contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based on the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By [3H]retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced [3H]retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the signal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I.     

Accessibility of the carbohydrate moiety of membrane-bound rhodopsin to enzymatic and chemical modification

Galactose was specifically inserted into the carbohydrate moiety of rhodopsin by incubating retinal disk membranes with UDP-galactose: N-acetylglucosamine galactosyltransferase. The stoichiometry of labeling ranged from 1.2 to 1.8 (average = 1.5) residues of galactose per molecule of rhodopsin, indicating that some or all of the oligosaccharide chains of membrane-bound rhodopsin are readily accessible to enzymatic modification. These modified membranes were treated with galactose oxidase to generate an aldehyde at the C-6 position of the inserted galactose units. The enzymatically-oxidized membranes were then reacted with dansyl hydrazide to yield a fluorescent hydrazone which is sufficiently stable to permit spectroscopic analysis. This procedure for the specific attachment of a spectroscopic probe should be applicable to a wide variety of membrane glycoproteins.     

Use of protein-mediated lipid exchange in the study of membrane-bound enzymes. The lipid dependence of glucose-6-phosphatase.

The ability of liver lipid-exchange proteins to introduce foreign phospholipids into microsomes was used in a study of the lipid dependence of glucose-6-phosphatase. Supplementation of intact rat liver and hepatoma microsomes with exogeneous aminophospholipids prevents the decline of glucose-6-phosphatase activity during incubation, whereas the introduction of exogeneous phosphatidylcholine has no protective effect. On the contrary with deoxycholate-disrupted hepatoma microsomes, introduction of additional phosphatidylcholine causes activation while phosphatidylethanolamine has only little effect. The results are explained by assuming that the transport unit and the catalytic moiety of the glucose-6-phosphatase system have different lipid requirements, the activity of the former protein depending mainly on phosphatidylethanolamine and phosphatidylserine and that of the catalytic protein depending on phosphatidylcholine. In deoxycholate-disrupted liver microsomes (in which both the glucose-6-phosphatase activity and the phosphatidylcholine content are much higher than in hepatoma microsomes) incubation with phosphatidylcholine and lipid-exchange proteins alters neither the phospholipid composition nor the enzyme activity. THis suggests that the diminished activity of glucose-6-phosphatase in hepatomas may be partly due to a low level of phosphatidylcholine.     

Different modes of proton translocation by sensory rhodopsin I.

The membrane-bound complex between sensory rhodopsin I (SRI) and its transducer HtrI forms the functional photoreceptor unit that allows transmission of light signals to the flagellar motor. Although being a photosensor, SRI, the mutant SRI-D76N and the HtrI-SRI complex can transport protons, as we demonstrate by using the sensitive and ion-specific black lipid membrane technique. SRI sustains an orange light-driven (one-photon-driven) outward proton transport which is enhanced by additional blue light (two-photon-driven). The vectoriality of the two-photon-driven transport could be reversed at neutral pH from the outward to the inward direction by switching the cut-off wavelength of the long wavelength light from 550 to 630 nm. The cut-off wavelength determining the reversal point decreases with decreasing pH. The currents could be enhanced by azide. A two-photon-driven inward proton transport by SRI-D76N (catalyzed by azide) and by the complex HtrI-SRI is demonstrated. The influence of pH and azide concentration on the rise and decay kinetics of the SRI380 intermediate is analyzed. The different modes of proton translocation of the SRI species are discussed on the basis of a general model of proton translocation of retinal proteins and in the context of signal transduction.