Location of two sulfhydryl groups in the rhodopsin molecule by use of the spin label technique

Sulfhydryl groups of membrane-bound rhodopsin are studied with the spin label technique by using five maleimide derivative probes of different lengths. Two sulfhydryl groups are titrated per molecule of rhodopsin. These groups are located in protected but probably different environments, less then 12 Å away from the aqueous phase. A distance of about 37 Å is measured between the two groups. These results are consistent with a model in which the two groups would be located close by the external surface of the protein but embedded within the membrane layer, the strong immobilization of the label molecules resulting from phospholipid-protein interactions.     

Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.     

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.     

Fluorescent and spin label probes of the environments of the sulfhydryl groups of porcine muscle adenylate kinase.

The environments of the two sulfhydryl groups of procine muscle adenylate kinase have been investigated by chemical modification reactions. The results indicate that the environments of the two-SH groups of procine muscle adenylate kinase are markedly different and that substrates induce conformational changes in the enzyme in the region of the sulfhydryl groups. The fluorogenic reagent 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-chloride) reacts specifically with the -SH groups of the enzyme at pH 7.9. One thiol group reacts with NBD-chloride approximately 40-fold faster than the other one, and the fast reacting group has been identified as Cys-25 in the amino acid sequence. The similarity of the rate of the more slowly reacting Cys-187 with NBD-chloride to that of glutathione with the same reagent is consistent with its location on the surface of the enzyme as determined by x-ray crystallography structure. The fast reacting Cys-25 in the interior of the structure can be approached by compounds such as NBD-chloride via a cleft. Reaction of Cys-25, presumably located close to the catalytic center, leads to complete inactivation of the enzyme. Substrates such as ATP, MgATP, and ADP which bind to the triphosphate subsite of the enzyme decrease the rate of reaction of Cys-25 by factors up to 3.5 but have only a small effect (approximately equal to 10%) on the reactivity of Cys-187. AMP, however, has a pronounced effect on the reactivity of Cys-187, the slowly reacting group. The multisubstrate analogue P-1, P-5-di-(adenosine-5)pentaphosphate (Ap-5A) decreases the rate of reaction of the fast reacting thiol group by a factor of 300. The behavior of Cys-25 toward NBD-chloride, i.e. super-reactivity in the absense of Ap-5A and slow reactivity in the presence of the multisubstrate inhibitor, was characteristic for both porcin and carp adenylate kinase. In the presence of Ap-5A adenylate kinase can be selectively modified at Cys-187; the introduction of the fluorescent NBD group at this position has no effect on enzymatic activity. A slow transfer of the NBD group occurs from the third groups to the epsilon-amino group of Lys-31. This transfer reaction is further evidence that the structure of adenylate kinase in dilute solution is similar to that of the crystalline enzyme since the x-ray data have shown that the sulfur of Cys-187 and the epsilon-nitrogen of Lys-31 are less than 4 A apart. The strongly fluorescent NBD-NH-enzyme possesses full activity and binds substrates as. cont'd     

The role of sulfhydryl groups in the bleaching and synthesis of rhodopsin

The condensation of retinene₁ with opsin to form rhodopsin is optimal at pH about 6, a pH which favors the condensation of retinene₁ with sulfhydryl rather than with amino groups. The synthesis of rhodopsin, though unaffected by the less powerful sulfhydryl reagents, monoiodoacetic acid and its amide, is inhibited completely by p-chloromercuribenzoate (PCMB). This inhibition is reversed in part by the addition of glutathione. PCMB does not attack rhodopsin itself, nor does it react with retinene₁. Its action in this system is confined to the —SH groups of opsin. Under some conditions the synthesis of rhodopsin is aided by the presence of such a sulfhydryl compound as glutathione, which helps to keep the —SH groups of opsin free and reduced. By means of the amperometric silver titration of Kolthoff and Harris, it is shown that sulfhydryl groups are liberated in the bleaching of rhodopsin, two such groups for each retinene₁ molecule that appears. This is true equally of rhodopsin from the retinas of cattle, frogs) and squid. The exposure of new sulfhydryl groups adds an important element to the growing evidence that relates the bleaching of rhodopsin to protein denaturation. The place of sulfhydryl groups in the structure of rhodopsin is still uncertain. They may be concerned directly in binding the chromophore to opsin; or alternatively they may furnish hydrogen atoms for some reductive change by which the chromophore is formed from retinene₁. In the amperometric silver titration, the bleaching of rhodopsin yields directly an electrical variation. This phenomenon may have some fundamental connection with the role of rhodopsin in visual excitation, and may provide a model of the excitation process in general.     

Evidence that the two free sulfhydryl groups of plasma fibronectin are in different local environments. Saturation-recovery electron spin resonance study.

Human plasma fibronectin is a dimer consisting of two subunits; each contains two cryptic thiol groups that were selectively labeled with an 15N,2H-maleimide spin label. Previous studies using conventional X-band electron spin resonance (ESR) methods showed that the spectrum of the labeled protein displays a single strongly immobilized component with an effective rotational correlation time of approximately 17 ns, suggesting that the physical environments of the two labeled sites per chain are indistinguishable. Here we have used saturation-recovery ESR to measure directly electron spin-lattice relaxation time (T1) of the labeled protein in solution at 27 degrees C. Interestingly, the time evolution of the signal was found to be biphasic, which was deconvoluted into two T1 values of 1.37 and 4.53 microseconds. Thus, the two spin-labeled sulfhydryl sites of plasma fibronectin (Fn), being similar in rates of rotational diffusion, differ by a factor of 3.2 in T1. Parallel experiments using various fibronectin fragments showed that the 1.37-microseconds component is associated with the label attached onto the thiol located in between the DNA-binding and the cell-binding domains, and the 4.53-microseconds component is associated with the label attached onto the thiol located within the carboxyl-terminal fibrin-binding domain. The data suggest that the saturation-recovery ESR is a useful method for differentiating multiple spin-labeled sites on macromolecules in which the labels undergo similar rates of rotational motion.     

Analysis of mobility of protein side chains by spin label technique

Five spin labeled derivatives of a neurotoxin from cobra venom were analyzed by the earlier suggested method. The procedure was adjusted to the complex motional behaviour of the label. Each protein derivative carried covalently bound spin label on different lysine residues. In two derivatives, at positions Lys44 and Lys46, the labels were strongly mobile, whereas for other three derivatives modified at Lys15, Lys25 and Lys26 the label was less mobile with respect to the protein molecule, which made possible determination of the rotational correlation time of the protein molecule (2.8±0.3 ns). The rotational correlation time was in good agreement with the calculated value for the rigid sphere of the corresponding molecular weight. On the basis of the estimate of the anisotropic motion degree, it was found from the order parameter S that the label mobility increases in the following series of lysine residues: Lys26, Lys25, Lys15, Lys46, and Lys44. From the analysis of positions of outer wide peaks in ESR spectra obtained by varying temperature and viscosity of the medium, we determined the parameters for computer simulation. The theoretical and experimental spectra were found to be in good agreement.Nomenclature rotational correlation time of the protein molecule - _l rotational correlation time of the spin label - 2A _Z , 2A the rigid limit distance between OWP and IWP, respectively, for ESR spectra of spin labeled proteins - 2, 2 the averaged limit distance between the OWP and IWP correspondingly mobile spin label to respect of protein moiety with; = - 2A',2A distance between OWP and IWP in the ESR spectra of spin labeled proteins for any T and media Abbreviations SL spin label - NT neurotoxin II from cobra venom - NT-SL-Lys44 neurotoxin spin labeled at Lys44 residue - OWP outer wide peaks in the immobilized ESR spectra - IWP inner wide peaks in the immobilized ESR spectra - WL the residual linewidth     

Characterization of sulphydryl groups of the mitochondrial phosphate translocator by a maleimide spin label

A maleimide spin label strongly inhibits the phosphate/H+ symporter of rat liver mitochondria. While inducing half-maximal inhibition of transport, the spin label reacts preferentially with the SH groups of the carrier, which are at least of two types. One type of SH group is localized close to the surface of the membrane and its environment does not significantly influence the mobility of the probe. The second type of SH group is buried in the membrane, is not accessible to ascorbate or chromium oxalate and its environment greatly restricts the motion of the probe.     

Biochemical aspects of the visual process XXVIII. Classification of sulfhydryl groups in rhodopsin and other photoreceptor membrane proteins

Reaction of isolated bovine rod outer segment membrane with radioactiveN-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule.On the basis of their reactivity towardsp-chloromercuribenzoate andp-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with thep-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and is probably located in the vicinity of the chromophoric center.The difference between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) orN-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of thep-chloromercuri-derivatives.     

Reactive sulfhydryl groups of the band 3 polypeptide from human erythroycte membranes. Location in the primary structure.

Human erythrocyte membranes contain a major transmembrane protein, known as Band 3, that is involved in anion transport. This protein contains a total of five reactive sulfhydryl groups, which can be assigned to either of two classes on the basis of their susceptibility to release from the membrane by trypsin. Two of the groups are located in the region COOH-terminal to the extracellular chymotrypsin-sensitive site of the protein and remain with a membrane-bound 55,000-dalton fragment generated by trypsin treatment. The three sulfhydryl groups NH2-terminal to the extracellular chymotrypsin site are released from the cytoplasmic surface of the membrane by trypsin. All three groups are present in a 20,000-dalton tryptic fragment of Band 3. Two of these groups are located very close to the sites of trypsin cleavage that generate the 20,000-dalton fragment. The third reactve group is probably located about 15,000-daltons from the most NH2-terminal sulfhydryl group. Two other well defined fragments of the protein do not contain reactive sulfhydryl groups. They are a 23,000-dalton fragment derived from the NH2-terminal end that is also released by trypsin from the cytoplasmic surface of the membrane and a 19,000-dalton membrane-bound region of the protein that is produced by treatment with chymotrypsin in ghosts. The 20,000-dalton tryptic fragment may, therefore, constitute a sulfhydryl-containing domain of the Band 3 protein.     

A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates

This paper suggests a simple modification of the Ellman procedure when used to measure accurate changes in sulfhydryl (-SH) content induced by reactive oxygen intermediates (ROI). This modification became necessary when we found that the standard technique did not produce time invariant results in the presence of ROI-generating systems. Cysteine (cys; 20–100 μM) in 20 mM imidazole buffer (pH 7.0) containing 1.0 mM EDTA was reacted with excess (0.2 mM) 5,5′-dithiobis(2-nitrobenzoic acid), DTNB. The absorbance of the product (p-nitrothiophenol anion) was recorded at 412 nm (A₄₁₂). This A₄₁₂ was stable for 60 min and gave a linear relationship with cys concentrations used. ROI were generated either by 0.01 U xanthine oxidase (XO) + 0.01–1.0 mM hypoxanthine (HX), 0.01–1.0 mM H₂O₂, or H₂O₂ + 100 μM FeSO₄. In the presence of ROI, A₄₁₂ decreased with time and its rate of decrease was dependent upon the concentration of components of the ROI-generating system. This time-dependent decrease in A₄₁₂ was prevented completely by the addition of 100 U of catalase (CAT). Therefore, we modified the DTNB method as follows: -SH groups were reacted with ROI for 30 min; this was followed by the addition of 100 U of CAT to scavenge the excess unreacted ROI before the addition of DTNB to generate the product. Using this modification the ROI-induced decrease in A₄₁₂ was stable with time and was linearly related to the cys concentration. We further tested the modified procedure using metallothionein (MT) as a substrate for the ROI-induced changes in -SH content. MT, at concentrations of 2.5, 5.0, and 7.5 μM, was treated with XO + 100 μM HX. Using the modified procedure, an average decrease (as compared to the untreated control) of 15, 22, and 33 μM in -SH content was observed consistently at the respective MT concentrations. However, without the modification in the procedure, these average decrease were 20, 38, and 51 μM, respectively and continued to further increase with time. These discrepancies could give rise to errors ranging from 28 to 35% or higher in determination of the ROI-induced decrease in the -SH groups of MT. This data suggests that scavenging the unreacted H₂O₂ with C prior to the addition of DTNB to the assay mixture gives a stable and accurate estimate of the ROI-induced oxidative damage to -SH groups.     

Oxidation of sulfhydryl groups to disulfides by sulfoxides.

Oxidation rates of SH groups in penicillamine, cysteine, and glutathione to the corresponding SS forms by dimethyl sulfoxide and other sulfoxides as a function of pH of the solvent and structure of reactants were measured by NMR spectroscopy. The observed second-order rate constant showed a biphasic pH dependence. A mechanism which rationalizes this result is proposed. These oxidations are proposed to have synthetic utility with biochemical implications.