Single Cysteinyl Residue Research Articles

Single residue within the antigen translocation complex TAP controls the epitope repertoire by stabilizing a receptive conformation

The recognition of virus infected or malignantly transformed cells by cytotoxic T lymphocytes critically depends on the transporter associated with antigen processing (TAP), which delivers proteasomal degradation products into the endoplasmic reticulum lumen for subsequent loading of major histocompatibility complex class I molecules. Here we have identified a single cysteinyl residue in the TAP complex that modulates peptide binding and translocation, thereby restricting the epitope repertoire. Cysteine 213 in human TAP2 was found to be part of a newly uncovered substrate-binding site crucial for peptide recognition. This residue contacts the peptide in the binding pocket in an orientated manner. The translocation complex can be reversibly inactivated by thiol modification of this cysteinyl residue. As part of an unexpected mechanism, this residue is crucial in complementing the binding pocket for a given subset of epitopes as well as in maintaining a substrate-receptive conformation of the translocation complex.

Block of Sodium Channels by Divalent Mercury: Role of Specific Cysteinyl Residues in the P-Loop Region

Divalent mercury (Hg 2+) blocked human skeletal Na + channels (hSkM1) in a stable dose-dependent manner ( K d = 0.96 μM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg 2+ block of hSkM1, and Hg 2+ block was also readily reversed by DTT. Both thimerosal and 2,2′-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg 2+ block of hSkM1. Y401C+E758C rat skeletal muscle Na + channels ( μ1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg 2+ ( K d = 18 μM). However, Y401C+E758C μ1 after reduction with DTT had a significantly higher sensitivity to Hg 2+ ( K d = 0.36 μM) than wild-type hSkM1. Mutants C753A μ1 ( K d = 8.47 μM) or C1521A μ1 ( K d = 8.63 μM) exhibited significantly lower sensitivity to Hg 2+ than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg 2+ block of the hSkM1 isoform. The heart Na + channel (hH1) was significantly more sensitive to low-dose Hg 2+ ( K d = 0.43 μM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance ( K d = 1.12 μM) to Hg 2+ than did wild-type hH1. In summary, Hg 2+ probably inhibits the muscle Na + channels at more than one cysteinyl residue in the Na + channel P-loop region. Hg 2+ exhibits a lower K d value (<1.23 μM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K d value (>8.47 μM) by forming sulfur-Hg + covalently. The heart Na + channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity ( K d < 0.43 μM) to Hg +, accounting for heart-specific high sensitivity to the divalent mercury.

Methanopyrus kandleri Glutamyl-tRNA Reductase

The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate.

Specific cardiolipin binding interferes with labeling of sulfhydryl residues in the adenosine diphosphate/adenosine triphosphate carrier protein from beef heart mitochondria.

The interaction of cardiolipin with the isolated ADP/ATP carrier protein from beef heart mitochondria has been studied by means of the unmasking of a single cysteinyl residue, Cys56, which accompanies the conformational transition of the protein [Leblanc, P., & Clauser, H, (1972) FEBS Lett. 23, 107-113]. The unmasking was monitored by using the static fluorescence of the sulfhydryl reagent N-(1-pyrenyl)maleimide (PYM). The rate of PYM binding that was observed after initiation of the conformational transition by ADP was drastically reduced in the presence of cardiolipin (CL). Phospholipids other than CL were much less effective. It can be shown that the conformational transition and the binding reaction are both affected by CL, although to varying extents. An enhancement of the rate of the ADP-dependent PYM binding was observed upon digestion of the protein bound phospholipid by phospholipase A2. The phospholipase treatment also led to an increased ADP-independent PYM binding, thus indicating that the ADP control of the carrier transition was gradually lost. The ADP control could be fully restored through the addition of CL, provided that the phospholipase incubation had been terminated after approximately 1 h. These results will be discussed in relation to an earlier report of tight cardiolipin binding [Beyer, K., & Klingenberg, M. (1985) Biochemistry 24, 3821-3826] and to current structural models of the ADP/ATP carrier protein.

Purification and Molecular Cloning of Arginine Kinase from Tail Muscle of Crayfish, Procambarus Clarkii.

We have purified adenosine triphosphate: arginine phosphotransferase (EC2-7-3-3, arginine kinase) and molecularly cloned the cDNA from the tail muscle of crayfish, Procambarus clarkii. An open reading frame encodes 357 amino acid residues with a calculated molecular mass of 40, 118 dalton. The predicted amino acid sequence shows 60% similarity with that of human creatine kinase B-subunit. The expression of a full-length cDNA in COS7 cells gave rise to a product of 40kDa and conferred the arginine kinase activity on mammalian cells. The amino acid sequence surrounding cys271 is well conserved as an active site for the catalytic function among a family of phosphagen kinase. We have found that chemical modification of a single cysteinyl residue of arginine kinase inhibits the enzymatic function and ATP protects the enzyme against this modification.

Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases.

Phosphatase inhibitor 2 was mutagenized and expressed in Escherichia coli to produce a protein with a single cysteinyl residue at position 129. The newly introduced sulfhydryl group was labeled with a maleimide derivative of coumarin (CPM). The resulting fluorescent inhibitor 2 molecule (CPM-I2) retains biological activity and binds to the catalytic subunit of type 1 phosphatase (PP1-C) with a Kd similar to the Ki of native I2 (2-3 nM). Fluorescence anisotropy data indicate that kinase FA (glycogen synthase kinase 3) does not dissociate the CPM-I2.PP1-C complex but rather causes a conformational change in the I2 molecule that is retained even after the CPM-I2 is displaced by an excess of native I2. The fluorescence data presented here also indicate that okadaic acid and I2 are competitive for binding to PP1-C, even after kinase FA treatment of the CPM-I2.PP1-C complex.

Modification of the adipocyte lipid binding protein by sulfhydryl reagents and analysis of the fatty acid binding domain

The adipocyte lipid binding protein (ALBP) is a member of a multigene family of low molecular weight proteins which stoichiometrically and saturably bind hydrophobic ligands and presumably facilitate intracellular lipid metabolism. To probe the structure-function relationship of the binding domain of ALBP, chemical modification has been employed. Modification of the two cysteinyl residues of ALBP (Cys1 and Cys117) with a variety of sulfhydryl reagents decreased the apparent affinity for oleic acid in the following order of effectiveness: methyl methanethiosulfonate much much less than p-(chloromercuri)benzenesulfonic acid less than N-ethylmaleimide (NEM) = 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Thiol titration of ALBP with DTNB in the presence of bound oleate resulted in the modification of a single cysteinyl residue. The oleate-protected cysteine was identified as Cys117 by modification with a combination of reversible (DTNB) and irreversible (NEM) sulfhydryl reagents in the presence or absence of saturating oleic acid. Cys117-NEM ALBP exhibited a large decrease in binding affinity while Cys1-NEM ALBP exhibited normal binding properties. Neither the modification of ALBP with NEM nor the addition of oleic acid had a significant effect on protein structure, as judged by circular dichroic analysis. These results suggest that Cys117 of ALBP resides in the ligand binding domain and that site-specific modification can be utilized to assess the conformational flexibility of the binding cavity.

Spectral properties of fluorescent derivatives of the oligomycin sensitivity conferring protein and analysis of their interaction with the F1 and F0 sectors of the mitochondrial ATPase complex.

In order to study the kinetics and the nature of the interactions between the oligomycin sensitivity conferring protein (OSCP) and the F0 and F1 sectors of the mitochondrial ATPase complex, fluorescent derivatives of OSCP, which are fully biologically active, have been prepared by reaction of OSCP with the following fluorescent thiol reagents: 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (Mal-ANS), N-(1-pyrenyl)maleimide (Mal-pyrene), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (Mal-coumarin), and fluorescein 5-maleimide (Mal-fluorescein). The preparation of these derivatives was based on the previous finding that the single cysteinyl residue of OSCP, Cys 118, can be covalently modified by alkylating reagents without loss of biological activity [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. For all fluorescent probes used, except Mal-pyrene and Mal-fluorescein, the emission spectra of conjugated OSCP were blue-shifted relative to those of the corresponding mercaptoethanol adducts, indicating that the fluorophores attached to Cys 118 were located in a hydrophobic pocket. These results were consistent with the high quantum yields and the increased fluorescence lifetimes of conjugated OSCP compared to mercaptoethanol adducts in aqueous buffer. They also fit with quenching data obtained with potassium iodide which showed that the fluorophore is shielded from the aqueous medium when it is attached to Cys 118 of OSCP. Especially noticeable was the wide half-width of the OSCP-acrylodan emission peak compared to that of mercaptoethanol-acrylodan.(ABSTRACT TRUNCATED AT 250 WORDS)

Frictional resistance to motions of bimane-labelled spinach calmodulin in response to ligand binding

The single cysteinyl residue 26 of spinach calmodulin was labelled with the thiol-specific bimane fluorescence probe. Following application of stoichiometric quantities of Ca 2+ or aluminum ions to the protein, temperature-dependent fluorescence changes (anisotropy, lifetime) could be monitored via the label. From these data the Y function could be constructed which, as a function of temperature, seems to consist of two linear regions which intersect at the critical temperature, T c. From the Y function the thermal coefficient, b( T), of the frictional resistance to fluorophore rotation could be determined. b( T) was dependent on the type and stoichiometry of the ligand(s) bound to calmodulin. Changes of the thermal coefficient apparently resulted in part from ligand-triggered structural pertubations transmitted over a considerable distance to calmodulin region I, the site of the fluorophore.

Ligand-triggered conformational perturbations elicit changes at the single cysteinyl residue of spinach calmodulin.

Following application of stoichiometric amounts of Ca2+ or specific partner peptides to spinach calmodulin, dynamic changes in the nanosecond range could be monitored at a strategically anchored fluorescence or spin probe. For these studies the single cysteinyl residue 26 of spinach calmodulin was labelled with a thiol-specific proxyl (i.e. 2,2,5,5-tetramethyl-1-pyrrolidinyl-oxyl) spin probe or with a bimane fluorescence probe. With Ca2+ and a specific ligand (mastoparan) present, fluorescence studies (anisotropy, lifetime) indicated that the rotational motion of the protein complex becomes slower relative to the motion of calmodulin in the absence of the specific ligand. The probe's attachment site 26 appears to reside in a fairly polar microenvironment as reported by a series of proxyl spin probes varying in label length. The rotational correlation time of the shortest spin probe markedly changed upon binding of a specific peptide to a calmodulin region distant from that of the monitoring spin probe. We interpret these observations as indicating that ligand-triggered conformational perturbations are eliciting specific responses at the cysteinyl residue 26 of spinach calmodulin.

A fluorescent derivative of the oligomycin-sensitivity conferring protein (acrylodan-OSCP). Evidence for polarity changes in the environment of CYS 118 of OSCP upon binding to mitochondrial F 1

The fluorescent probe, 6-acryloyl-2-dimethylaminonaphtalene (acrylodan) was reacted with the oligomycin-sensitivity conferring protein (OSCP). Acrylodan bound covalently to the single cysteinyl residue of the protein. Acrylodan-OSCP was fully competent in conferring oligomycin sensitivity to the mitochondrial F 0-F 1 ATPase complex. The fluorescence emission peak of acrylodan-OSCP was blue-shifted compared to that of an acrylodan-mercaptoethanol adduct, which means that acrylodan experiences a hydrophobic environment in OSCP. Binding of acrylodan-OSCP to the isolated F 1 was accompanied by a red shift of fluorescence. It was achieved in less than 1 s at 25°C. The titration curve revealed one high affinity OSCP binding site per F 1. Acrylodan-OSCP appears to be an interesting tool for studying the dynamics of structural changes within the mitochondrial ATPase complex.

Cold-labile hemolysin produced by limited proteolysis of theta-toxin from Clostridium perfringens.

A nicked toxin whose hemolytic activity is temperature dependent was obtained by limited proteolysis of theta-toxin (Mr 54,000) with subtilisin. The nicked toxin (C theta) is a complex of two fragments: the N-terminal fragment (Mr 15,000) with basic isoelectric point and the C-terminal fragment (Mr 39,000) with the single cysteinyl residue of the toxin whose reduced form is essential for the hemolytic activity. C theta hemolyzes erythrocytes only at temperatures above 25 degrees C, whereas the native toxin hemolyzes them even at 10 degrees C. At temperatures below 25 degrees C, C theta does not hemolyze them although it does bind to membrane cholesterol and although no distinct difference was observed between the secondary structure of C theta and that of native toxin. It was found that C theta binds to the cells only in a reversible manner at low temperature, while the native one binds irreversibly to the cells within 10 min, which explains the cold lability of C theta on hemolysis. The structural basis of the cold lability was discussed through comparison of C theta with another nicked derivative of theta-toxin that was also obtained.

Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes.

In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit. Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit. In other work [Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta. As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 m-NH 4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(±3)nm (mean value ± standard deviation: n = 105).

Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether.

To align the four cyanogen bromide peptides of Neurospora tyrosinase whose amino acid sequences were reported in the preceding paper, suitable methionine-containing overlap peptides were isolated. The required peptides were obtained by tryptic, peptic, and thermolytic digestion of the unmodified protein and of the maleylated derivative. From the partial sequence information of these peptides and a cyanogen bromide overlap peptide, the four cyanogen bromide fragments were aligned in the order CB3-CB1-CB4-CB2. These data establish Neurospora tyrosinase as a single-chain protein of 407 amino acids with a molecular weight of 46,000. The single cysteinyl residue 94 was found to be covalently linked via a thioether bridge to histidyl residue 96. The chemical nature of this unusual structure was elucidated by physicochemical analysis of peptides obtained from in vivo 35S, [2,5-3H]histidine, and [5-3H]histidine-labeled Neurospora tyrosinase.

Studies on the alkali light chains of vertebrate skeletal muscle myosin. Effect of tyrosyl modification on the ability to reassociate to heavy chains.

The structures of the alkali light chain subunits A1 and A2 have been studied by examining the effect of the conformationally sensitive reagent tetranitromethane, which reacts specifically with tyrosyl residues. Whereas reaction in the presence of 6 M guanidine hydrochloride results in modification of the three tyrosyl residues of both these light chains, only two tyrosyl residues are exposed to the reagent in the native conformations of these proteins. By gel chromatography of the CNBr-cleaved chains it was demonstrated that the two reactive tyrosyls are those located in the CB-1 and CB-3 segments and that these tyrosyl residues are modified simultaneously and not sequentially. The unreactive tyrosyl residue is in the CB-6 segment and is separated by two residues from the single cysteinyl residue of these chains. It is found that the modified light chains cannot be made to reassociate with the heavy chains by the NH4Cl hybridization procedure of Wagner and Weeds [J. Mol. Biol. 109, 455-470 (1977)] or by the thermal hybridization procedure [Burke and Sivaramakrishnan (1981) Biochemistry 20, 5908-5913]. Furthermore, reduction of the nitrotyrosyl groups to aminotyrosyl residues by sodium dithionite does not restore this effect. The data suggest that regions of the light chains at CB-1 and CB-3 are involved in the association to the heavy chains.

Immobilization of proteins as a tool for studying primary structure around their cysteinyl residues

The primary structure around the single cysteinyl residue of chicken pepsin was investigated by binding the protein via this residue to an insoluble carrier. Carriers stable towards reagents used for the fragmentation of proteins and sequence analysis were prepared by coupling a spacer arm to polyN-hydroxymethyl acrylamide using a thioether bond that is potentially cleavable by mercuric ions (1). Phenacyl bromide group, attached to the free end of the spacer, reacted rapidly and specifically with the cysteinyl residue of chicken pepsin. Up to 300 mg of the enzyme were bound to 1 g of carrier.The polymer-bound protein was cleaved by trypsin or by cyanogen bromide or by a sequence of both. Fragments of 40-120 amino acid residues, depending on the method of cleavage, remained attached to the polymer through the cysteinyl residue. The compositions and partial sequences of these fragments revealed that the cysteinyl residue is located within or in the vicinity of a loop in the molecule formed by a disulfide bond.

The role of the cysteinyl and one of the tryptophyl residues in the neurotoxic action of suberitine.

The selective photooxidation of the single cysteinyl residue and one of the 3 tryptophyl residues of suberitine has been performed by irradiation using crystal violet and proflavine respectively as photosensitizers. Crystal violet and the protein form a 1:1 complex with a consequent partial inhibition of the neurotoxic activity. The latter is completely abolished by specific photooxidation of cysteine which is probably involved in the active site of the protein. The modification of the tryptophyl residue induces a large loss of the activity as a consequence of a photoinduced extensive denaturation of suberitine.

Primary structure of yeast cytochrome c peroxidase : I. Chemical characterization of the polypeptide chain and of tryptic and chymotryptic peptides

The amino acid composition of yeast cytochrome c peroxidase was determined and calculated assuming that the enzyme contained one protoheme per molecule. On the basis of the amino acid composition and heme content the minimum M r was calculated to be 35,235. Gel electrophoresis in the presence of sodium dodecyl sulfate indicated the presence of a single polypeptide chain with a M r of approximately 33,000. A single cysteinyl residue present in the molecule was shown to be resistant against reaction with iodoacetic acid in the native form of both holo- and apo-enzymes, but readily modified with the reagent in the denatured form. Automated Edman degradation yielded an aminoterminal sequence of 11 residues beginning with threonine. Twenty-eight tryptic and 47 chymotryptic peptides were isolated from the carboxymethylated apoprotein and subjected to the sequence analysis by the dansyl-Edman method. The results with these peptides confirmed and extended the amino- and carboxyl-terminal sequences and in addition provided a partial sequence covering approximately 90% of the polypeptide chain.

Modification of the reactive sulfhydryl of bacterial luciferase with spin-labeled maleimides

Luciferase from the luminous marine bacterium Beneckea harveyi has been modified at a single cysteinyl residue by reaction with an equimolar amount of a spin-labeled maleimide: 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (I) or 3-(3-maleimidopropyl carbamoyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (II). The resulting enzymes modified with compound I or with compound II were inactive. The apparent second-order rate constants for reaction of luciferase with I and II at pH 7.0, 25 °C, were (5.7 ± 0.1) × 10 4 and (1.1 ± 0.2) × 10 5 m −1 min −1, respectively. Examination of the labeled luciferases by electron spin resonance spectroscopy indicated that the polarity of the region occupied by the nitroxide, and thus presumably of the active center, is similar to that of 2-propanol and that the reactive sulfhydryl resides in a cleft at least 17 Å in length.