Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.

The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.     

Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.     

Ethoxyformylation of bovine growth hormone

Reactivity of histidines in bovine growth hormone towards ethoxyformic anhydride was investigated and localization in the molecule of two kinetically distinguishable classes was achieved, a slow class including only histidine residue 169 (k = 0.180 min-1) and a fast one composed of histidines 19 and 21 (k = 0.900 min-1). Total ethoxyformylation of bovine growth hormone brought about a complete loss of its capacity to compete with 125I-labelled hormone for rat-liver binding sites, but modification of approximately half of the fast histidine group was enough to produce an important decrease in this capacity. Circular dichroism studies indicated no significant changes in protein conformation with all three histidine residues modified. Practically full binding capacity was restored when these residues were regenerated by treatment with hydroxylamine. These results suggest that one or both of the fast reacting histidine residues are involved in bovine growth hormone binding to its specific receptors.     

Molecular properties of the oxytocin/bovine neurophysin biosynthetic precursor. Studies using a semisynthetic precursor

An oxytocin/bovine neurophysin I biosynthetic precursor, [N epsilon-diacetimidyl-30,71, des-His106]pro-OT/BNPI, was synthesized from a synthetic oxytocinyl peptide, 1/2Cys-Tyr-Ile-Gln-Asn-1/2Cys-Pro-Leu-Gly-Gly-Lys-Arg, and native neurophysin by chemical semisynthesis. The semisynthetic precursor contains the entire sequence of the biosynthetic precursor deduced from the complementary DNA structure except for omission of the carboxyl-terminal histidine residue. The covalent structure of the semisynthetic product was verified by amino acid analysis and amino-terminal analysis. Analytical affinity chromatography was employed to evaluate noncovalent binding properties of the precursor. The precursor does not bind significantly to immobilized Met-Tyr-Phe, a hormone binding site ligand. In contrast, the acetimidated precursor binds to immobilized bovine neurophysin II, with a 13-fold higher affinity than does acetimidated neurophysin itself. When a hormonal ligand, [Lys8]vasopressin, was added to the elution buffer at the concentration of 0.1 mM so that a major portion of the immobilized BNPII was liganded, the affinity between the immobilized liganded BNPII and the precursor was enhanced 8-fold and approached the affinity for the liganded (bovine neurophysin I-immobilized BNPII) interaction. The data imply that the precursor can self-associate and that this self-association is closely related to that of liganded neurophysin. The tripeptide affinity matrix data argue that, in the precursor, the ligand binding site of the neurophysin domain is occupied intramolecularly by the hormone domain. The data verify the view that both the self-association surface and hormone binding site are established upon precursor folding. A disulfide stability analysis showed the resistance, to disulfide interchange by dithiothreitol, of semisynthetic precursor but not of neurophysin, as judged by protein association and peptide ligand binding activities, respectively. The results argue that the molecular structure of the precursor is established upon precursor folding and before enzymatic processing that produces mature hormone and neurophysin.     

Effects of singlet oxygen on the extracellular matrix protein collagen: oxidation of the collagen crosslink histidinohydroxylysinonorleucine and histidine

The reaction of singlet oxygen, a putative agent of skin photodamage, with the dermal collagen crosslink histidinohydroxylysinonorleucine (HHL) and its precursor histidine is reported. Reaction studies were performed with both purified HHL and bovine dermal tissue. We demonstrate that singlet oxygen can selectively oxidize HHL and histidine amino acid residues in dermal tissue and that intermediate oxidation products of histidine lead to new crosslink products. A novel mechanism for crosslink formation was proposed to involve nucleophilic addition to a transient imidazolone intermediate formed from singlet oxygen oxidation of the histidine imidazole moiety. The implication for such adduct formation and histidine oxidation in collagen proteins is the expression of aberrant collagen crosslinks, perturbation of the dermal collagen function, and hence an altered dermal state.     

The primary structure of the nonspecific lipid transfer protein (sterol carrier protein 2) from bovine liver

The primary structure of the nonspecific lipid transfer protein (sterol carrier protein 2) from bovine liver has been determined. The protein consists of a single polypeptide chain of 121 amino acid residues with serine as the amino-terminal and alanine as the carboxy-terminal residue. The protein contains one single cysteine and tryptophan residue and lacks tyrosine, histidine and arginine.     

Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.     

Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen

A stable nonreducible trifunctional cross-linking amino acid has been isolated from mature bovine skin collagen fibrils. Previous cross-link peptide isolations and amino acid analyses indicate the compound has properties identical with those of hydroxyaldolhistidine. Its newly proposed structure was verified using fast atom bombardment mass spectrometry, and 1H and 13C nuclear magnetic resonance. The data indicated that the cross-link consists of the prosthetic groups from one residue each of histidine, hydroxylysine, and lysine. The 1H and 13C nuclear magnetic resonance data indicated that imidazole C-2 of histidine is linked to C-6 of norleucine (epsilon-deaminated lysine residue) which in turn is linked to the C-6 amino group of hydroxylysine. Based on the trivial names for other cross-linking residues found in collagen and elastin it was given the name histidinohydroxylysinonorleucine. In vitro incubation studies for up to 24 weeks, in aqueous solution at physiological pH and ionic strength, using 6-month-old bovine embryo skin demonstrated a one-to-one stoichiometric relationship between the disappearance of the labile reducible bifunctional cross-link dehydrohydroxylysinonorleucine and the appearance of histidinohydroxylsinonorleucine. These results can partially explain the previously observed disappearance of dehydrohydroxylysinonorleucine with chronological age.     

On the domain structure of antithrombin III. Tentative localization of the heparin binding region using 1H NMR spectroscopy

The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by 1H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053] at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.     

1H nuclear magnetic resonance study of the histidine residues of insulin

Insulin has proved difficult to study by nuclear magnetic resonance spectroscopy because of its complex aggregation behaviour in solution and its insolubility between pH 4 and 7. Now for the first time it has been possible to assign the ¹H nuclear magnetic resonances of the H-2 histidine protons of residues B5 and B10 of bovine 2 Zn insulin and Zn-free insulin, and the B5 and A8 residues of hagfish insulin. As expected, the addition of Zn to Zn-free insulin causes virtually no change in the chemical shift or the rate of H-D exchange of the H-2 proton of histidine B5, which is not involved in Zn binding in the 2 Zn insulin hexamer. The rate of H-D exchange of the H-2 proton of histidine B10 is decreased markedly on Zn binding at this residue, but the chemical shift of the resonance remains virtually constant owing to the balancing of an upfield ring current shift of the ordered histidine residues by a downfield shift due to electron withdrawal from the ring nitrogen by the Zn binding.     

Proton nuclear magnetic resonance studies of histidine residues in rat and other rodent pancreatic ribonucleases. Effects of pH and inhibitors

Proton nuclear magnetic resonance spectra of the histidine residues in bovine and rat ribonuclease have been compared. The changes in chemical shift on titration and on binding of cytidine-3′-monophosphate and cytidine-2′-monophosphate have been followed. In the presence of the cytidine derivatives the spectra of both enzymes resemble each other more than those of the free enzymes. With these inhibitors, two histidines in rat ribonuclease exhibit the same pK values and shifts as the active site residues histidine 12 and 119 in the bovine enzyme. Their pK values in the inhibitor-free rat enzyme are about 0.4 higher than in the beef enzyme, which can be explained by the substitution at the entrance of the active site cleft of arginine 39 in the beef enzyme by serine in the rat enzyme. Rat ribonuclease contains one histidine with a rather high pK value of 7.6. The cytidine derivatives affect its chemical shift in exactly the same way as the shift of histidine 48 in bovine ribonuclease. The high pK value of this residue in rat ribonuclease can be explained by assuming a strong hydrogen bridge with glutamic acid 16. The other two histidines in rat ribonuclease have rather low pK values of 6.1 and 6.3. The histidine with a pK value of 6.3 has been assigned to position 105 and that with a pK value of 6.1 to position 73.The closer resemblance of the active sites of bovine and rat ribonuclease in the presence of inhibitors than in the inhibitor-free enzymes makes the concept of induced fit interesting from an evolutionary point of view.The characteristic downfield shift of the protonated form of histidine 119 in the complexes of bovine and rat ribonuclease with cytidine-3′-monophosphate is not observed with uridine-3′-monophosphate, suggesting non-identical binding of these pyrimidine nucleotides.Some preliminary results on the nuclear magnetic resonance properties of the histidine residues in coypu and chinchilla pancreatic ribonuclease have been obtained.     

Bisphenol derivative of allysine for high-performance liquid chromatographic analysis of allysine residue of proteins

Allysine is the most important precursor of physiologically essential cross-links formation in collagen and elastin and is formed by enzymatic oxidative deamination of lysine residues. Because it is a highly reactive aldehyde, many cross-linking amino acid residues may arise from its reaction with other allysine residues or lysine or even histidine residues. We purified and isolated an allysine bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxyphenylpentane (ACPP), from the reaction products of phenol and allysine residue of bovine ligamentum nuchae by acid hydrolysis in 6 M HCl. The structure of ACPP was verified by UV, fast atom bombardment-MS, ¹H- and ¹³C-nuclear magnetic resonance spectroscopies. The optimal reaction condition for ACPP synthesis accompanied by hydrolysis of such proteins was investigated and an ion-paired high-performance liquid chromatographic method for determination of allysine as ACPP was also developed.     

A new method of coupling proteins to insoluble polymers

The A chain isolated from biologically active human thrombin is thirteen residues shorter from the amino terminus than the A chain isolated from bovine thrombin. Automated Edman degradation of the reduced, alkylated A chain permitted the direct identification of 34 out of the 36 residues and established all tryptic overlaps. The NH₂-terminal residue is threonine. This residue is homologous to threonine-14 of the A chain of bovine thrombin. Human thrombin A chain has one half cystine linking it to the larger B chain. Methionine, histidine, valine and tryptophan are not present. There are nine acid residues (Glu, Asp) but none of their respective amides.     

Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin

Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains a conserved post-translationally modified histidine residue known as diphthamide. Diphthamide is a unique site of ADP-ribosylation by diphtheria toxin (DT), which is responsible for cell killing. In this report, we describe the construction of DT-resistant HeLa cell lines by engineering the toxin-resistant form of its specific substrate, protein elongation factor-2. Using site-specific mutagenesis of the histidine precursor of diphthamide, the histidine residue of codon 715 in human EF-2 cDNA was substituted with one of four amino acid residue codons: leucine, methionine, asparagine or glutamine. Mutant EF-2s were subcloned into a pCMVexSVneo expression vector, transfected into HeLa cells, and DT-resistant cell clones were isolated. The protective effect of mutant EF-2s against cell killing by DT, after exposing all four mutant strains derived from HeLa cells to different concentrations of the toxin (5-20 ng/mL) was demonstrated by: (1) the normal morphological appearance of the cells; (2) their unaffected or slightly slower growth rates; (3) their undisturbed electrophoretic DNA profiles whose integrity was virtually preserved. Mutant cell strains showed also considerable levels of resistance to very high concentrations of DT, in that they maintained slower but consistent rates of cell growth. It was hence concluded that despite its strict conservation and unique modification, the diphthamide histidine appears not to be essential to the function of human EF-2 in protein synthesis. In addition, DT-resistant HeLa cell clones should prove valuable hosts for various DT gene-containing vectors that express the toxin intracellularly.     

Protein splicing of a Pyrococcus abyssi intein with a C-terminal glutamine

Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond. Most reported inteins have a C-terminal asparagine residue, and it has been shown that cyclization of this residue is coupled to peptide bond cleavage between the intein and C-extein. We show that the intein interrupting the DNA polymerase II DP2 subunit in Pyrococcus abyssi, which has a C-terminal glutamine, is capable of facilitating protein splicing. Substitution of an asparagine for the C-terminal glutamine moderately improves the rate and extent of protein splicing. However, substitution of an alanine for the penultimate histidine residue, with either asparagine or glutamine in the C-terminal position, prevents protein splicing and facilitates cleavage at the intein N terminus. The intein facilitates in vitro protein splicing only at temperatures above 30 degrees C and can be purified as a nonspliced precursor. This temperature dependence has enabled us to characterize the optimal in vitro splicing conditions and determine the rate constants for splicing as a function of temperature.     

Proton-magnetic-resonance spectroscopic study of the histidine residues of bovine alpha-lactalbumin.

A study of the three histidine residues of bovine alpha-lactalbumin has been made using proton magnetic resonance (PMR) spectroscopy in order to obtain information on their environments in the protein and thereby to test in part the previously proposed structure. PMR titration curves are obtained for the H-4 resonances using difference spectroscopy and for the H-2 resonances and the 1-H-2-H exchange rates of the H-2 protons have been measured. The assignment of resonances to particular histidine residues is achieved by utilising their selective reaction with iodoacetate in conjunction with a PMR study of the carboxymethylation of alpha-N-acetyl-L-histidine. The H-2 and H-4 resonances labelled 1, 2 and 3 starting from the downfield end of the spectrum are assigned to histidine residues 107, 68 and 32 respectively. Their apparent pK values at low ionic strength and 20 degrees C are 5.78, 6.49 and 6.51 respectively. The experimental results on two histidine residues are consistent with the predictions of the proposed structure, which indicate that histidine-68 is an external residue and histidine-32 is partially buried and in the vicinity of aromatic residues. The experimental data on histidine 107 can also be rationalised with less certainty in terms of the proposed structure, which indicates a partially buried residue that may be involved in hydrogen bonding.     

A novel form of the polypeptide PHI isolated in high yield from bovine upper intestine. Relationships to other peptides of the glucagon-secretin family

A novel form of the polypeptide termed PHI (peptide HI with N-terminal histidine and C-terminal isoleucine amide) has been isolated from bovine upper intestine. This bovine peptide was obtained in a 40 times higher yield than the corresponding polypeptide isolated from porcine intestine. Bovine PHI is, like porcine PHI, composed of 27 amino acid residues. The complete amino acid sequence of the bovine peptide is His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser- Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence differs from porcine PHI at position 10 and from human PHI at positions 10, 12 and 27. The amino acid residue exchange between porcine and bovine PHI makes the latter more similar to the vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), glucagon and the growth-hormone-releasing factor (GRF).     

Effect of Methylene Blue on glutamate and reduced glutathione of rabbit erythrocytes.

The C-2 proton of one histidine residue in bovine erythrocyte superoxide dismutase is shown to be particularly labile. This residue is identified by tritiation, protein digestion and subsequent peptide 'mapping' as histidine-41. A half-life for the exchange of histidine C-2 1H for 2H in 2H2O as solvent, at pD 8.1 and 40 degrees C, is estimated as approx. 9.2h, by 1H nuclear-magnetic-resonance spectroscopy.     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.     

Isolation and characterization of bovine pancreastatin

Bovine pancreastatin, a 47 amino acid residue peptide, was isolated from the pancreas and the pituitary gland using a chemical method which detects its C-terminal glycine amide structure. The complete amino acid sequence of the pancreatic peptide is 74% homologous to that of porcine pancreastatin and is identical to bovine chromogranin A-(248-294), as deduced from its cDNA sequence. The sequence of the first 28 amino-terminal residues of the pituitary peptide was determined to be identical to the corresponding sequence of the pancreatic peptide. Since the pituitary peptide also contains the C-terminal glycine amide, it is therefore likely to be identical in structure to the pancreatic peptide. Thus, we conclude that bovine chromogranin A is the precursor of bovine pancreastatin. Synthetic bovine pancreastatin inhibited pancreatic exocrine secretion in a similar manner to porcine pancreastatin.