Histidine Residue In Position Research Articles

Locating Pb2+ and Zn2+ in Zinc Finger-Like Peptides Using Mass Spectrometry

The binding preferences of Pb(2+)and Zn(2+) in doubly charged complexes with zinc finger-like 12-residue peptides (Pep), [Mn(Pep-2(n-1)H)](2+) have been explored using tandem mass spectrometry. The peptides were synthesized strategically by blocking the N-terminus with an acetyl group and with four cysteine and/or histidine residues in positions 2, 5, 8, and 11, arranged in different motifs: CCHH, CHCH, and CCCC. The MS(2) spectra of the Pb(2+) and Zn(2+) complexes show multiple losses of water and a single methane loss and these provide a sensitive method for locating the metal dication and so elucidating its coordination. The elimination of a methane molecule indicated the position of the metal at the Cys2 residue. Whereas lead was observed to preferentially bind to cysteine residues, zinc was found to primarily bind to histidine residues and secondarily to cysteine residues. Preferential binding of lead to cysteine is preserved in the complexes with more than one Pb(2+). Key to the mechanism of the loss of water and methane is the metal dication withdrawing electrons from the proximal amidic nitrogen. This acidic nitrogen loses its hydrogen to an amidic oxygen situated four atoms away leading to formation of a five-member ring and the elimination of water.

Histidine Residues in the Peptide d-Lys6-GnRH: Potential for Copolymerization in Polymeric Nanoparticles

Poly(ethylcyanoacrylate) (PECA) nanoparticles containing the bioactive d-Lys(6)-GnRH were manufactured by an in situ interfacial polymerization process using a w/o-microemulsion template containing the peptide in the dispersed aqueous pseudophase of the microemulsion. Polymeric nanoparticles were characterized using PCS, RP-HPLC (bulk level) and MALDI TOF mass spectrometry (molecular level). The peptide d-Lys(6)-GnRH was reactive with the alkylcyanoacrylate monomer, resulting in some of the peptide copolymerizing with the monomer. MALDI TOF/TOF (tandem) analysis revealed that the histidine residue in position 2 of d-Lys(6)-GnRH interacts covalently in the polymerization process. A reaction mechanism for this nucleophilic interference is suggested. The copolymerization reaction appeared to occur within seconds after the addition of the monomer to the microemulsion. The surface charge of resulting nanoparticles was less negative (-3 mV) compared with the zeta potential of empty nanoparticles (-27.5 mV). The copolymerization yielded high entrapment rates of 95 +/- 4% of peptide, but showed limited release ( approximately 11%) of free peptide over 5 days. A separate experiment demonstrated that the addition of d-Lys(6)-GnRH to preformed empty PECA nanoparticles (ex situ) also yielded fractions of copolymerized peptide suggesting a certain proportion of polymer remains available for copolymerization possibly through an unzipping depolymerization/repolymerization process. Therefore, the reactivity of histidine residues in bioactives needs to be considered whenever using the bioactive in situ or ex situ with polymeric PECA nanoparticles.

A new CE‐ESI‐MS method for the detection of stable hemoglobin acetaldehyde adducts, potential biomarkers of alcohol abuse

A new CE-ESI-MS method was developed to provide a simple way to study changes to hemoglobin (HbA) induced by acetaldehyde (Ach) in vitro. Instrumental parameters were univariately optimized in order to maximize the sensitivity of the CE-ESI-MS method. The electrophoretic separations were carried out in poly-E323-coated capillaries using 60 mM formic acid raised to pH 3.0 with ammonia and containing 5% 2-propanol while the sheath liquid, 2-propanol/water (30:70) with 0.1% formic acid, was delivered at 1.0 microL/min through a coaxial sheath flow electrospray interface. The HbA was incubated with Ach for intervals up to 24 h at concentration varying in the window 0.2-20 mM. Four stable Ach-hemoglobin adducts in the hemoglobin tryptic digest were observed at the submillimolar Ach concentration and characterized by MS/MS experiments: although the alpha and beta N-amino terminal modifications were expected, the two internal ones arising, respectively, from the condensation of Ach molecules on the histidine residue in position 4 in alpha4 (i.e. the fourth peptide after tryptic digestion of alpha chain starting from amino terminal) and on the asparagine residue in position 2 in beta3, were identified for the first time. During the in vitro experiments higher concentrations of Ach were also used; however, it was not possible to identify any other stable modification of hemoglobin. Interestingly, those stable modifications are the only ones in vivo identified in the hemoglobin of moderate alcohol drinkers.

Designed High Affinity Cu2+-Binding α-Helical Foldamer

The design, synthesis, and characterization of a folded high-affinity metal-binding peptide is described. Based on the previously described folded peptide NTH-18, in which an alpha-helix was constrained through two disulfide bonds to a C-terminal extension of noncanonical secondary structure, a peptide (1) was designed to contain two histidine residues in positions 3 and 7. Air oxidation of 1 led to the formation of peptide 2, which contained two intramolecular disulfide bonds. The presence of the two histidines significantly destabilized the alpha-helical structure of 2 when compared to NTH-18. However, CD spectroscopy revealed that the addition of certain transition metal ions allowed the reformation of a stable alpha-helix. CD, NMR, and EPR spectroscopy as well as MALDI-TOF mass spectrometry indicated that 2 bound to Cu2+ to form a 1:1 complex via the imidazoles of the two histidine side chains. A glycine displacement assay revealed a dissociation constant for this complex of 5 nM at pH 8, which is the lowest reported value for a designed Cu2+-binding peptide. This peptide displayed more than 100-fold selectivity for Cu2+ over Zn2+, Ni2+, and Co2+. The 1.05 A crystal structure of the Cu(II)-complex of 2 revealed a square-pyramidal coordination geometry and confirmed that 2 bound to copper in an alpha-helical conformation via its two histidine side chains. The high affinity metal binding of peptide 2 demonstrates that metals can be used for the selective nucleation of alpha-helices.

PH-dependent modulation of Kv1.3 inactivation: role of His399.

The Kv1.3 K(+) channel lacks N-type inactivation, but during prolonged depolarized periods it inactivates via the slow (P/C type) mechanism. It bears a titratable histidine residue in position 399 (equivalent of Shaker 449), a site known to influence the rate of slow inactivation. As opposed to several other voltage-gated K(+) channels, slow inactivation of Kv1.3 is slowed when extracellular pH (pH(o)) is lowered under physiological conditions. Our findings are as follows. First, when His399 was mutated to a lysine, arginine, leucine, valine or tyrosine, extracellular acidification (pH 5.5) accelerated inactivation reminiscent of other Kv channels. Second, inactivation of the wild-type channel was accelerated by low pH(o) when the ionic strength of the external solution was raised. Inactivation of the H399K mutant was also accelerated by high ionic strength at pH 7.35 but not the inactivation of H399L. Third, after the external application of blocking barium ions, recovery of the wild-type current during washout was slower in low pH(o). Fourth, the dissociation rate of Ba(2+) was pH insensitive for both H399K and H399L. Furthermore, Ba(2+) dissociation rates were equal for H399K and the wild type at pH 5.5 and were equal for H399L and the wild type at pH 7.35. These observations support a model in which the electric field of the protonated histidines creates a potential barrier for potassium ions just outside the external mouth of the pore that hinders their exit from the binding site controlling inactivation. In Kv1.3, this effect overrides the generally observed speeding of slow inactivation when pH(o) is reduced.

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel.

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an alpha-helix stretch (residues 5-12). This alpha-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an alpha-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional 1H nuclear magnetic resonance and show that 1) the proline residue does not disturb the alpha-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an alpha-helix running from residues 5 to 12, two antiparallel stretches of beta-sheet (residues 15-20 and 23-27) connected by a type I' beta-turn, and three disulfide bridges connecting the alpha-helix to the beta-sheet.

Coordination of the [2Fe-2S] cluster in wild type and molecular variants of Clostridium pasteurianum ferredoxin, investigated by ESEEM spectroscopy.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56, and 60. This pattern is unique, and a combination of site-directed mutagenesis and spectroscopy is therefore being implemented to identify the ligands of the [2Fe-2S] cluster. The possible involvement of ligands other than cysteine in some molecular variants of this ferredoxin has been considered, histidines being likely candidates. Therefore, the three histidine residues in positions 6, 7, and 90 of the amino acid sequence have been individually and collectively replaced by alanine or valine. The mutated ferredoxins have been purified and were all found to contain [2Fe-2S] clusters of which the UV-visible absorption spectra were identical to that of the wild-type protein. The H6A/H7A/ H90A triply mutated ferredoxin was further characterized by EPR and by ESEEM spectroscopy and was found to differ only marginally from the wild-type protein. The ESEEM spectra of wild-type ferredoxin displayed weak 14N hyperfine interactions at the three principal g-factors of the [2Fe-2S] center. The estimated 14N coupling constants (Aiso = 0.6 MHz; e2qQ approximately 3.3 MHz) indicate that the ESEEM effect is most likely due to 14N from the polypeptide backbone. 2H2O ESEEM spectra showed that the [2Fe-2S] cluster is accessible for exchange with solvent deuterons. ESEEM spectra of the previously characterized C24A and C14A/C24A variants have been recorded and were also found to be very similar to those of the wild-type protein. There was no evidence for coordination of the [2Fe-2S] cluster by [14N]histidine or other 14N nuclei, in either wild-type or mutant forms of the ferredoxin. By these criteria, the environment of the [2Fe-2S] center is not distinguishable from those in plant-type ferredoxins. Non-cysteinyl coordination most probably occurs only in the C14A/C24A variant, which contains no more than three cysteine residues. The data shown here indicate that the fourth ligand of the [2Fe-2S] cluster is neither a histidine residue nor another nitrogenous ligand. The possibility of oxygenic coordination for this molecular variant is discussed.

Role of histidine 95 on pH gating of the cardiac gap junction protein connexin43.

We have studied the role of histidine 95 (H95) on the pH gating of the cardiac gap junction protein connexin43 (Cx43). Wild-type and mutant rat cardiac Cx43 channels were expressed in antisense-injected Xenopus oocytes. Junctional conductance was measured using the dual voltage-clamp technique, and intracellular acidification was induced by superfusion with a sodium acetate-containing solution balanced at a pH of 6.2. H95 was substituted by other amino acids by use of oligonucleotide-directed site-specific mutagenesis. Replacing H95 for the hydrophobic residues methionine or phenylalanine, for the charged basic residue arginine, or for the noncharged residue glutamine (H95Q) yielded nonfunctional channels. Functional expression of H95Q was rescued by placing a histidine residue in position 93 (H95Q-L93H), 94 (H95Q-A94H), or 97 (H95Q-F97H) but not in position 96. Further experiments showed that replacing H95 with either aspartate (an acidic residue) or tyrosine (a polar uncharged residue) led to the expression of functional channels with a reduced susceptibility to acidification-induced uncoupling, whereas lysine (a basic residue) was more susceptible to uncoupling than the wild-type protein. The susceptibility to acidification-induced uncoupling was enhanced for the H95Q-A94H mutant when compared with the wild-type mutant, but it was significantly reduced when histidine was placed at position 93 (H95Q-L93H). Our data indicate that a properly placed histidine residue is an important structural element for functional expression as well as for pH regulation of Cx43. The results suggest that the importance of H95 on pH gating may be associated with a possible protonation of this residue on acidification of the intracellular environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Polypeptides. Part 16. Synthesis and biological activity of α-aza-analogues of luliberin with high antagonist activity

Analogues of luliberin (luteinising hormone-releasing hormone) were synthesised where the glycine residue in position 10 was replaced by either azaglycine (–NH–NH–CO–) or aza-alanine (–NH–NMe–CO–), the histidine residue in position 2 was either omitted, or replaced by D-phenylalanine or D-tryptophan, and the glycine residue in position 6 was substituted either by D-phenylalanine or D-tryptophan. These compounds were evaluated for their ability to prevent ovulation induced by luliberin in androgen-sterilised constant-oestrus rats. Compounds with the azaglycine residue in position 10 and other modifications in positions 2 and 6 showed good antagonist activity, whereas aza-alanine replacement in position 10 together with modifications in position 2 resulted in inactive compounds. The most potent analogue, [2-D-Phe-6-D-Phe-10-Azgly]-luliberin, completely inhibited ovulation induced by luliberin (0.5 µg/rat) at a dose of 15 µg/rat.

Isolation, Amino Acid Sequence and Copper(II)-binding Properties of Peptide (1–24) of Dog Serum Albumin

Abstract The NH2-terminal peptide fragment (1–24) of dog serum albumin was obtained by a limited peptic hydrolysis of whole albumin. The peptide fragment was isolated and subsequently purified to homogeneity by trichloroacetic acid fractionation, Sephadex gel filtration, and high voltage electrophoresis at pH 2.0. The purity of the peptide was established by 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) analysis of the NH2-terminal residue, by amino acid analysis and by cellulose acetate electrophoresis at pH 8.6. The complete sequence, obtained by dansyl-Edman degradation on the whole peptide and by characterization of its tryptic peptides, was shown to be Glu-Ala-Tyr-Lys-Ser-Glu-Ile-Ala-His-Arg-Tyr-Asn-Asp-Leu-Gly-Glu-Glu-His-Phe-Arg-Gly-Leu-Val-Leu, which gave a molecular weight of 2847. The amide charge was obtained by amino acid analysis of residues released after enzymic digestion of the tryptic peptide. It was proposed earlier that in dog serum albumin the lack of specificity of Cu(II) binding was due to the absence of a histidine residue in position 3 (Appleton, D. W., and Sarkar, B. (1971) J. Biol. Chem. 246, 5040–5046). The present results show that a single base change in the albumin gene has replaced the important histidine-3 residue, critical to the binding of Cu(II), with a tyrosine residue. The homology between the NH2-terminal peptide from dog serum albumin and those from bovine, rat and human is discussed. To elucidate the role of these first 24 residues in the observed binding phenomenon of the intact dog serum albumin molecule, Cu(II)-binding properties of the peptide fragment were investigated by spectrophotometric and proton displacement studies. In both the intact protein and the 1–24 peptide fragment, the first equivalent of Cu(II) is distributed between at least two sites, indicating a nonspecific binding. This partitioning of the first equivalent of Cu(II) probably occurs between the NH2-terminal binding site and another site containing a histidine residue. Thus, a mutation in the codon specifying residue 3 in the dog peptide has resulted in an altered Cu(II) binding site of reduced specificity.

KINETICS AND MECHANISMS OF REACTIONS CATALYZED BY PANCREATIC RIBONUCLEASE

A kinetic study has been made of the ribonuclease-catalyzed hydrolyses of three cyclic nucleotides, cytidine-2′,3′-phosphate, uridine-2′,3′-phosphate, and N6,O5′-diacetyl cytidine-2′,3′-phosphate. Rates were measured at pH values ranging from 6 to 8.5. The variation of the kinetic parameters with pH showed that the free enzyme possesses two active groups, having pK values of 5.4 and 7.25. When the enzyme–substrate complex is formed, the pK values of the groups are increased to 6.6 and 8.4. The pK values identify these groups as imidazole groups and show that two histidine residues are present at the active site. Since both increase in pK on complex formation, it is concluded that the acid imidazole group binds the substrate, but that the basic imidazole group cannot be concerned in substrate binding and must function only in the hydrolytic step. The results indicate that the pyrimidine base is concerned in the hydrolytic step and not solely in binding, as had been postulated. It is concluded from all of the evidence that four specific sites are present at the active center of the enzyme; three are involved in binding and one in catalysis. It is proposed that the active site of ribonuclease is composed of: the histidine residue in position 12, which catalyzes the hydrolytic step; the histidine residue in position 119, which binds the 2′-ribose oxygen atom in the substrate; the lysine residue in position 41, which binds the phosphate group or anion; and the aspartic acid residue in position 121, which binds the nitrogen atom at N1in the pyrimidine base. A mechanism for enzyme–substrate complex formation and subsequent hydrolysis is proposed.

Neurospora crassa Cytochrome c: II. CHYMOTRYPTIC PEPTIDES, TRYPTIC PEPTIDES, CYANOGEN BROMIDE PEPTIDES, AND THE COMPLETE AMINO ACID SEQUENCE

Abstract The complete amino acid sequence of Neurospora crassa cytochrome c has been determined. The molecule consists of a single polypeptide chain containing 107 residues. The NH2-terminal residue is glycine, and the COOH-terminal residue is alanine. There are 39 amino acid differences between the cytochromes of N. crassa and those of the yeast Saccharomyces cerevisiae, whereas there are 43 amino acid differences between the cytochromes c of Neurospora and human heart. For all of the presently known cytochromes c, the number of amino acids which occupy the same position is reduced to 39. N. crassa cytochrome c has only 2 histidine residues in positions 18 and 33, whereas tuna fish cytochrome has 2 histidine residues in positions 18 and 26. It is suggested that the histidine residue in position 18 contributes the fifth ligand to the heme iron, and that the sixth ligand in cytochrome c is not formed with histidine or with another nitrogenous group, but with an unknown ligand.

The catalytic nature of insulin

T HE PIJRPOSE of this communication is to relate structure in insulin alld chymotrypsin to thr enzymatic and hormonal activities of both compounds. Recognition of the amphibolic ilroperties of both proteins began when we noticed reselnblances in the behavior of chymotrypsin and trypsin to that of insulin in rat diaphragln [I]. This is illustrated in the first two figures. Figure 1 shows that chymotrypsin, applied to intact rat diaphragm at a concentration corresponding to 0.5 unit of insulin per ml., stimulates the accumulation of the nonmetabolized sugar 3-O-methyl glllcose and of L-proline to a greater extent than does insulin. Most of this insulin-like activity is lost when the enzymaticall? inactive diisopropyl fluorophosphate derivative of chymotrypsin is employed. Figure 2 shows the result of a 10 minute incubation of rat diaphragms in glucose. Trypsin favors the conLersion of glucose IO glycogcn, whereas the diisopropylfluorophosphate derivative of the enzyme is again ineffective. Chymotrypsin also stinlulates glycogen synthesis [I], although not to as great an extent as does trypsin; chymotrypsin acts Illore like insulin in encouraging substrate transport. The inslulin-like activity of chymotr-ypsin w-as confirmed by Dailey et al. I_‘] using the same concentrations of the enzyme. They also reco,qnized that enzyme concentrations corresponding to physiologic concentrations of insulin are without effect. Because diisopropylfluorophosphate phosphorylates the serine at the active site of these enzyn~es, in which a histidine-serine inter action fat-ms the basis of the catalytic lnechanism [,?+I], OII~ attention was drawn to the two speciesillvariant histidine residues in positions B 5 and B IO, and the two species-invariant serine residues in positions B 9 and A 12 of insulin. Since histidine at the acti\.e sites of trypsin and chymotrypsin Ilrust be brought close to the reactive scrine in order to effect catalysis, we pointed out I/ 1 that there is proximity of a serine and an unionized histidinc in insulin: serine B 9 is adjacent to histidine B 10 in all nlanltnalian insulins of known structure. These similarities, and the ubiquity of histidine at the active sites of enzymes, ted IIS to suspect that insulin might be endowed with proteolytic activity. This proved to be the cast; insulin is capable of catalyzing the hl-drolysis of a variety of proteins [:C;]. The lower record in Figure 3 traces the time course of the hydrolysis of native myoglobin catalyzed by 3.84 X IO G M insulin as followed in the pH-stat. For comparison, the upper record shows the same reaction catalyzed by an equimolar alnolmt of trypsin. The insulin-like behavior of trypsin and thymotrypsin 011 the one hand, and the proteolytlc activity of insulin on the other, suggest the involvement of catalytically active groups in the functions of the hormone. The recent elucidation of the complete amino acid sequence of chyinotrypsin [(i], its insulin-like activity, and the state of our knowledge of its catalytic mechanisln 171 made the use of this enzyme as an insulin model attractive [8]. The irreversible inactivation of chymotrypsin by L-l-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) results from the alkylation of an active site histidine in position 57 of the B chain of the enzyme [+ I I ]. Chynlotrypsin inactivated in this way was administered intraperitoneally to fed, alloxan-diabetic rats. Blood sugar studies (Fig. 4) reveal this chynlotrypsin derivative, as well as the DIP derivative. to be ineffective hypoglycelnic ageljts a:: cornpared with the native enzyme. The hypoglycemic action of chymotrypsill thus depends on the active site serine residue which diisopropylfluorophosphatc phosphorylates, and on the active site histidine, which TPCK alkylates. These results appear to establish identity of the enzymatically and hypoglycemically active sites in chymotrypsin and in the homologous protein, trypsin IS].