Affinity labeling of a tyrosine residue in the ATP binding site of the recA protein from Escherichia coli with 5'-p-fluorosulfonylbenzoyladenosine

We have covalently modified the recA protein from Escherichia coli with the adenine nucleotide analog 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA). The rate at which the protein is modified shows a sigmoidal dependence on the concentration of 5'-FSBA suggesting that binding of the analog is characterized by positive cooperativity. Covalent modification of the protein results in irreversible inactivation of its single-stranded DNA-dependent ATPase activity such that 100% inactivation is achieved when 25% of the enzyme monomers have been modified. Attachment of 5'-FSBA is specific for the ATP-binding site of recA protein as judged by the following criteria: (i) attachment of the affinity label to the protein appears to saturate at 1 mol of 5'-FSBA/mol of protein; (ii) binding of 5'-FSBA to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine-5'-O-(thiotriphosphate), ADP, UTP, and GTP, but not by adenosine; (iii) attachment of 5'-FSBA to the protein occurs at a single site as determined by high pressure liquid chromatography peptide separation. Following trypsin digestion of recA protein that had been covalently modified with [3H]5'-FSBA we isolated a single labeled peptide (T31) containing the exclusive site of 5'-FSBA attachment. A secondary proteolytic digestion was performed on both 5'-FSBA modified T31 and unmodified T31 using Staphylococcus aureus V8 protease, and by comparison of the amino acid compositions of the resulting peptides we identified Tyr-264 as the exclusive site of 5'-FSBA attachment in recA protein.     

Covalent modification of the recA protein from Escherichia coli with the photoaffinity label 8-azidoadenosine 5'-triphosphate

We have covalently modified the recA protein from Escherichia coli with the photoaffinity ATP analog 8-azido-[alpha-32P]ATP (N3-ATP). Covalent attachment of N3-ATP to recA protein is dependent on native protein conformation and is shown to be specific for the site of ATP hydrolysis by the following criteria. (i) Binding of the probe to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine 5'-O-(thiotriphosphate), ADP, and UTP, but not by adenosine; (ii) N3-ATP is efficiently hydrolyzed by recA protein in the presence of single-stranded DNA; (iii) labeling of recA protein occurs at a single site as judged by two-dimensional thin-layer peptide mapping and high-performance liquid chromatography peptide separation. We have purified and identified a tryptic fragment, spanning amino acid residues 257-280, which contains the primary site of attachment of N3-ATP. This peptide is likely to be contained within the ATP hydrolytic site of recA protein.     

Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r.

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.     

Evidence for nucleotide-mediated changes in the domain structure of the recA protein of Escherichia coli

We have used limited trypsin digestion as a means of investigating changes in the structural properties of recA protein accompanying the binding of different nucleoside triphosphates. The levels of four partial digestion products are greatly increased in digests of recA protein complexed with dTTP, dATP, ATP, or the ATP analogue adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). These bands (22, 19, and 17.5 kilodaltons) are absent or present at reduced levels in digests of recA protein alone. Unlike these nucleotides, all of which bind tightly to recA protein, nucleotides and analogues that bind poorly produce little or no change in the digestion pattern of recA protein. We have compared the rates of fragment accumulation in the presence of dTTP and show a saturable dependence on nucleotide concentration. Binding of single-stranded DNA to recA protein does not alter the pattern of digestion products compared to protein alone, and the digestion pattern of recA protein-DNA-ATP gamma S ternary complexes is similar to that of uncomplexed enzyme. We have used monoclonal antibody binding, high-performance liquid chromatography separation of peptides, and amino acid composition analyses to localize the regions of recA protein which are altered in their susceptibility to trypsin when nucleoside triphosphates are present. The results of these analyses indicate that the fragments arise from trypsin cutting at two or more sites near the middle of the primary sequence. These cleavage sites are more than 80-110 residues away from the site of photoaffinity labeling by 8-N3ATP (Tyr-264). Our results suggest that, in the presence of certain nucleotides, recA protein is organized into two stable structural domains.     

Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue

1. The 3'-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase has been tested. 2. In the dark 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3. Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the alpha-subunit and an approximately 12000-Mr component. 4. Photolabeling of the alpha-subunit by N3-ATP irreversibly inactivates (Na+ + K+)-ATPase. 5. Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6. Mg2+, in concentrations required for photoinactivation, protects (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7. It is assumed that a conformational change of the ATP-binding site of (Na+ + K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.     

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.     

Activation of ATPase activity of simian virus 40 large T antigen by the covalent affinity analog of ATP, fluorosulfonylbenzoyl 5''-adenosine.

Fluorosulfonylbenzoyl 5'-adenosine (FSBA) bound to one site in simian virus 40 large T antigen (T) and covalently modified greater than 95% of the molecules in a complete reaction. This analog for ATP specifically cross-links to the Mg-phosphate pocket in ATP-binding sites. Cyanogen bromide cleavage and tryptic digestion of [14C]FSBA-labeled protein, paired with T-specific monoclonal antibody analyses, were used to map the site in T to a tryptic peptide just C terminal to the PAb204 epitope. The location of the FSBA linkage was consistent with the predicted tertiary structure of the ATP-binding region in T described previously (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Binding of FSBA to T was cooperative, implying an interaction between two binding sites. This could occur if the protein formed a dimer, and it is known that the ATPase activity is associated with a dimeric T. Most interesting was the activation of the ATPase when up to 50% of T was bound by the analog. The effect was also produced by preincubation with millimolar concentrations of ATP or the nonhydrolyzable analog gamma beta-methylene 5'-adenosine diphosphate at elevated temperatures. When greater than 50% of T was modified by FSBA, the ATPase was inhibited as the analog cross-linked to the second, previously activated, binding site. These data support a dual function for the one ATP-binding site in T as both regulatory and catalytic.     

Identification of a novel autophosphorylation site (P4) on the epidermal growth factor receptor.

Three major autophosphorylation sites are located near the C-terminus of the epidermal growth factor receptor, but a fourth site is repeatedly detected. We report here the purification and sequencing of a tryptic peptide containing this site, Tyr-1086. Furthermore, we demonstrate that additional phosphopeptides are observed following both partial digestion and overdigestion. Finally, we show that Tyr-1086 can be phosphorylated in intact cells.     

Identification of amino acid residues modified by two ATP analogs in Bacillus subtilis glutamine synthetase.

Bacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP), each one containing either Mg2+ or Mn2+. The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm. The 8-N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm. A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91. The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B. subtilis GS were His-186, His-187, and Trp-424. These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits. The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B. subtilis GS. Since the same amino acids were labeled when B. subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B. subtilis GS binding Mn(2+)-ATP and that binding Mg(2+)-ATP was detected by affinity labeling with ATP analogs.     

Photoaffinity labeling of the thymidine triphosphate binding domain in Escherichia coli DNA polymerase I: identification of histidine-881 as the site of cross-linking

Using the technique of ultraviolet-mediated cross-linking of substrate deoxynucleoside triphosphates (dNTPs) to their acceptor site [Abraham, K. I., & Modak, M. J. (1984) Biochemistry 23, 1176-1182], we have labeled the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) with [alpha-32P]dTTP. Covalent cross-linking of [alpha-32P]dTTP to the Klenow fragment is shown to be at the substrate binding site by the following criteria: (a) the cross-linking reaction requires dTTP in its metal chelate form; (b) dTTP is readily competed out by other dNTPs as well as by substrate binding site directed reagents; (c) labeling with dTTP occurs at a single site as judged by peptide mapping. Under optimal conditions, a modification of approximately 20% of the enzyme was achieved. Following tryptic digestion of the [alpha-32P]dTTP-labeled Klenow fragment, reverse-phase high-performance liquid chromatography demonstrated that 80% of the radioactivity was contained within a single peptide. The amino acid composition and sequence of this peptide identified it as the peptide spanning amino acid residues 876-890 in the primary sequence of E. coli Pol I. Chymotrypsin and Staphylococcus aureus V8 protease digestion of the labeled tryptic peptide in each case yielded a single smaller fragment that was radioactive. Amino acid analysis and sequencing of these smaller peptides further narrowed the dTTP cross-linking site to within the region spanning residues 876-883. We concluded that histidine-881 is the primary attachment site for dTTP in E. coli DNA Pol I, since during amino acid sequencing analysis of all three radioactive peptides loss of the histidine residue at the expected cycle is observed.     

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[alpha-32P]ATP

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[alpha-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 microM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 microM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A-Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-beta-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.     

Selective fluorescent labeling at the α-amino group of bovine pancreatic trypsin inhibitor

Experimental investigation of protein structure and dynamics by spectroscopic methods using external probes requires attachment of a probe to a well-defined site and preparation of pure samples. Measurements of efficiency of nonradiative excitation energy transfer can yield very detailed information about the structure of proteins, provided that two different probes are selectively attached to well-defined sites. We have used specific protection of ε-amino groups using tert-butylazidoformate at high pH for covalent attachment of the fluorescent probe 2-naphthoxyacetic acid at the α-amino group of bovine pancreatic trypsin inhibitor (BPTI). The product is a chromatoraphically homogenous protein derivative that contains the probe at a dye to protein ratio of 1:1, specifically located at the N-terminus, and and that retains its full biological activity. The HPLC tryptic peptide map of BPTI has been analyzed, and all the peptide fragments have been identified. Analysis of tryptic fragments of the labled BPTI derivative showed that it was selectively labeled at the N-terminal amino acid. The probe absorbs in the 310–325-nm range, which is spectrally distinct from the absorption of the protein, and has a monoexponetial fluorescence decay. These and other charactristics make this probe a good energy donor in transfer-efficiency measurements.     

Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.     

The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.     

Affinity labeling of rabbit muscle fructose-1,6-bisphosphate aldolase with 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

Aldolase contains one tight binding site and one weak binding site per subunit for ATP [Kasprzak, A. and Kochman, M. (1980) Eur. J. Biochem. 104, 443-450]. The reaction of the ATP analog 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit aldolase A results in linear inactivation of enzyme with respect to covalent linkage of fluorescent label. The enzyme is completely protected against modification in the presence of saturating covalent binding (k2 = 0.033 min-1) is preceded by a fast reversible binding step (Ki = 6.8 mM). Chemical modification of aldolase leads to formation of stable N epsilon (4-carboxybenzenesulfonyl-lysine (Cbs-Lys) and O-(4-carboxybenzenesulfonyl-tyrosine (Cbs-Tyr) derivatives. Almost all Cbs-Lys was found in the N-terminal CNBr peptide (CN-1), whereas Cbs-Tyr was present both in the N-terminal (CN-1) and C-terminal (CN-2) peptide. From carboxypeptidase digestion and tryptic peptide analysis, Cbs-Lys was localized in position 107, a small part of Cbs-Tyr was detected in position 84, and the majority of Cbs-Tyr was found in the C-terminal position Tyr-363. We conclude that the covalent binding of the ATP analog occurs at the mononucleotide tight-binding site of aldolase and is associated with modification of Lys-107 and Tyr-363. This conclusion is based on the measurements of enzymatic activity loss as a function of ATP analog incorporation as well as on previous data. It is postulated that Lys-107, which is the C-6 phosphate binding site for fructose-1,6-P2, is in close proximity to the functionally important Tyr-363. The rather small extent of modification of Tyr-84 (0.15 mol/subunit), is due either to nonspecific protein modification or labeling of the weak mononucleotide binding site.     

Immunologically active and structurally similar fragments of protein A from Staphylococcus aureus.

To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc-part of human IgG were isolated by affinity chromatography on IgG-Sepharose 4B and purified by ion-exchange chromatography on phosphocellulose. From a partial tryptic digest of pure protein A at 30 degrees C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein-A-containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A. All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc-reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.     

Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP. Its interaction with Na,K-ATPase and kinases.

The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described. At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase. In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes. Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities. Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: [formula: see text] From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue. At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM. At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner. Inactivation results in the incorporation of [alpha-32P]8-CNS8-ATP into the catalytic alpha-subunit of the enzyme. Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase. The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes.     

Isolation of glycopeptides containing O-linked oligosaccharides by lectin affinity chromatography on jacalin-agarose

Jacalin is a lectin which has high specificity and affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides. Here, it is shown that this lectin can be used for isolation of glycopeptides bearing O-linked oligosaccharides. Peptides produced by digestion of reduced and carboxamidomethylated human plasminogen or of bovine protein Z were chromatographed on a column of jacalin-agarose. Reverse-phase high-performance liquid chromatography revealed that two peptides from plasminogen and one from protein Z were eluted from the jacalin-agarose column by alpha-methylgalactopyranoside. Amino acid sequence and compositional analysis showed that both of the peptides from plasminogen consisted of residues 330-357 and that the single peptide from protein Z represented residues 385-396. These sequences contain the single known site of attachment of O-linked oligosaccharides to these proteins. The present analysis suggested that there may be a fraction of plasminogen with two sites of O-linked glycosylation. The two tryptic peptides isolated from plasminogen represented the same segment of the protein but sequence analysis showed that one peptide was modified only at Thr346, the known site of glycosylation, and the other peptide contained a modification of Ser339 as well. Results of the present study indicate that lectin affinity chromatography using jacalin-agarose can be a useful technique for isolating glycopeptides containing O-linked oligosaccharides and thereby localizing sites of attachment of these oligosaccharides.     

A detailed examination of the iodination of beta-galactosidase: stoichiometric inactivation by nonspecific iodination

The incorporation of 125I, using lactoperoxidase, and the subsequent inactivation of beta-galactosidase in the period when incorporation and inactivation were stoichiometric were investigated in detail. The high pressure liquid chromatographic (HPLC) radioactive profiles of the tryptic peptides of samples taken in the stoichiometric period showed that, although two labelled peptides predominated, there were other labelled peptides. The predominating peptides were shown to be the mono- and di-iodinated forms of the peptide containing Tyr-253. This confirmed the result of an earlier study, but quantitation showed that this iodination accounted for only 15-18% of the total. To show that the other labelled peptides in the HPLC profiles were not merely oxidized or partially digested forms of the peptide containing Tyr-253, two experiments were carried out. In one of the experiments, two of the other labelled peptides were isolated and identified as iodinated forms of the peptide containing Tyr-285 (5-7% of the incorporation). In the other experiment, four additional labelled fractions from the HPLC eluate were treated further with trypsin. No further digestion was observed and thus these peptides did not result from incomplete digestion of the sequence containing Tyr-253. Overall, these results show that, although the incorporation of 125I was stoichiometric with inactivation, no single Tyr was responsible for the inactivation as was tentatively suggested previously. The competitive inhibitor isopropyl-beta-D-thiogalactopyranoside (IPTG) was effective in reducing the rates of inactivation of the enzyme and incorporation of 125I, but the same peptides were labelled in the presence of IPTG as in its absence.     

The nucleotide-binding site of HisP, a membrane protein of the histidine permease. Identification of amino acid residues photoaffinity labeled by 8-azido-ATP

The periplasmic histidine transport system (permease) of Escherichia coli and Salmonella typhimurium is composed of a soluble, histidine-binding receptor located in the periplasm and a complex of three membrane-bound proteins of which one, HisP, was shown previously to bind ATP. These permeases are energized by ATP. HisP is a member of a family of membrane transport proteins which is conserved in all periplasmic permeases and is presumed to be involved in coupling the energy of ATP to periplasmic transport. In this paper the nature of the ATP-binding site of HisP has been explored by identification of some of the residues that come into contact with ATP. HisP was derivatized with 8-azido-ATP (N3ATP). Both the underivatized and the derivatized forms of HisP were solubilized, purified, and digested with trypsin. The resulting tryptic peptides were resolved by high pressure liquid chromatography, and peptides modified by N3ATP were isolated and sequenced. Two peptides, X and Z, spanning amino acid residues 16-23 and 31-45, were found to contain sites of N3ATP attachment at His19 and Ser41, respectively. Both peptides are close to the amino-terminal end of HisP; peptide Z is located in one of the well conserved regions comprising the nucleotide-binding consensus motifs of the energy-coupling components of these permeases. These consensus motifs are found in many purine nucleotide-binding proteins. The relationship between the location of these residues and the overall structure of the ATP-binding site is discussed.