Histidine C2 Protons Research Articles

Motional dynamics of the catalytic loop in OMP synthase.

In de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotic acid and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). The known three-dimensional structure of the dimeric enzyme from Salmonella typhimurium is similar to that of other Type I phosphoribosyltransferases (nucleotide synthases) with a solvent-exposed active site atop a Rossman-type nucleotide binding fold. The three-dimensional structure of an enzyme-inhibitor complex [Henriksen et al. (1996) Biochemistry 35, 3803-3809] indicates that one of the two identical solvent-exposed loops can descend to cover the active site of the adjacent subunit of the dimeric enzyme. Catalytically essential residues are known to reside on this loop. In the present work, sensitivity toward limited proteolysis by trypsin confirms that the loop is solvent-exposed. Protection by PRPP and, to a lesser extent, by OMP demonstrates the existence of a second, trypsin-inaccessible, loop position. Two-dimensional 1H-15N NMR relaxation experiments on [alpha-15N]histidine-labeled WT OPRTase yielded backbone 15N T1 and T2 relaxation times and 15N[1H] NOE for His-105 (a loop residue) that are characteristic of small peptides. These results document that the surface loop is highly flexible in the unliganded enzyme. Addition of a hydrolytically stable PRPP analogue to the enzyme resulted in a significant reduction of His-105 peak intensity, indicating a dramatic change in the dynamic properties of the loop backbone in the analogue-ligated enzyme. 1H NMR titrations on histidine C2 protons, coupled with 1H and 31P titrations monitoring the C1H and 5-phosphate PRPP resonances, allowed the quantitation of the rates of loop movement during product release, and relate protein motion to enzymatic catalysis. These results suggest that loop opening and PRPP release is a two-step process, whose overall rate is partially rate-limiting in the reverse pyrophosphorolysis reaction.

Interaction of apocytochrome c and derived polypeptide fragments with sodium dodecyl sulfate micelles monitored by photochemically induced dynamic nuclear polarization proton NMR and fluorescence spectroscopy

The topology of apocytochrome c, the heme-free precursor of the mitochondrial protein cytochrome c, was investigated in a lipid-associated form. For this purpose photochemically induced dynamic nuclear polarization 1H nuclear magnetic resonance (CIDNP 1H NMR) spectroscopy and quenching of tryptophan and tyrosine fluorescence by acrylamide were applied to an apocytochrome c-sodium dodecyl sulfate (SDS) micellar system. A pH titration of the chemical shifts of the histidine C2 proton resonances of apocytochrome c, using conventional 1H NMR, yielded pK(a)'s of 5.9 +/- 0.1 and 6.2 +/- 0.1, which were assigned to histidine-18 and -33 and histidine-26, respectively. In the presence of SDS micelles an average pK(a) of 8.1 +/- 0.1 was obtained for all histidine C2 protons. Photo-CIDNP enhancements of the histidine, tryptophan, and tyrosine residues, contained in the intact apocytochrome c and in chemically and enzymatically prepared fragments of the precursor, were reduced in the presence of SDS micelles. Similarly, the quenching of the tryptophan fluorescence of the polypeptides by acrylamide was diminished in the presence of SDS. These results indicate the aromatic residues studied are localized in the interface of the SDS micelle.

1H-NMR study on the interactions of human serum albumin with free fatty acid.

Binding of free fatty acid (FFA) to human serum albumin (HSA) was studied by 1H-NMR spectroscopy. Addition of FFA to defatted HSA at a mole ratio (FFA/HSA) up to 4 caused a small change in the NMR spectrum of HSA. The integrated intensity of sharp signals of the histidine C2 proton region of HSA decreased as the mole ratio was increased from 0 to 4 for both medium chain (lauric acid) and long chain (palmitic acid, stearic acid, and oleic acid) FFA's. By contrast, when the mole ratio was increased above 4, several histidine C2 proton signals coalesced and sharpened. Therefore, the HSA molecule appears to have a different conformation on binding with more than 4 FFA molecules, which allows increased local motions of HSA. By analyzing the NMR difference spectra of HSA with various amounts of FFA, the conformational change of HSA was investigated in more detail. The difference spectrum between [HSA + 2FFA] and [HSA + FFA] was almost the same as the difference spectrum between [HSA + FFA] and [HSA], which suggests that one primary site binds a pair of FFA molecules. These results are consistent with those of a spectroscopic study with polyene fatty acids (Berde, C.B., et al. (1979) J. Biol. Chem. 254, 391-400). The existence of a bimolecular complex of FFA molecules in aqueous solution may facilitate this type of binding. Similarly, it was found that the third and fourth FFA molecules were bound to a secondary site on HSA, because the difference spectrum between [HSA + 4FFA] and [HSA + 3FFA] was nearly equal to the difference spectrum between [HSA + 3FFA] and [HSA + 2FFA]. Further addition of FFA resulted in a drastic spectral change of HSA. The NMR difference spectrum between HSA solutions with perdeuterated FFA and those with undeuterated FFA gave the 1H-NMR spectra of FFA molecules bound to HSA. Titration of FFA revealed that, in the binding to the primary site of HSA, the carboxyl group of FFA is tightly bound to the protein, whereas the methyl group is not so firmly bound. In contrast, in the binding to low affinity sites, the methyl group is bound to HSA as tightly as other portions of the molecule.

Nuclear Magnetic Resonance Study on the Microenvironments of Histidine Residues of Ribonuclease T<sub>1</sub> and Carboxymethylated Ribonuclease T<sub>1</sub><xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

The 270-MHz 1H NMR spectra and fluorescence of ribonuclease T1 and carboxymethylated ribonuclease T1 were measured in aqueous solution. Histidine C4 proton resonances were assigned to individual residues. From the pH dependences of the chemical shifts of histidine C2 and C4 protons, the pKa values of histidine residues were obtained by the non-linear least-squares method. The hydrogen leads to deuterium exchange rates of histidine C2 protons were determined as a measure of the accessibility of histidine residues to the solvent. Each histidine residue of ribonuclease T1 was found to interact with a carboxylate group of an aspartic or glutamic acid residue; in particular, His-40 was shown to interact with Glu-58. Upon carboxymethylation of Glu-58, His-92 and His-27 are more shielded from the solvent while His-40 remains exposed to the solvent. The 67.9-MHz 13C NMR spectra were measured for the 13C-enriched preparation of carboxymethylated ribonuclease T1. From the pH dependence of 13C chemical shift, the pKa value of the carboxymethylated Glu-58 was found to be unusually low, suggesting the formation of an ionic or hydrogen bond between this carboxymethyl group and a positively charged group, possibly of Arg-77.

Effects of coenzyme binding on histidine residues of Lactobacillus casei dihydrofolate reductase.

The effects of coenzyme binding on the seven histidine C2 proton resonances of Lactobacillus casei dihydrofolate reductase have been determined. Binary complexes containing NADP+, NADPH, and their hypoxanthine, thionicotinamide, and acetylpyridine analogues, together with ternary complexes containing the inhibitors trimethoprim or methotrexate, have been examined. Four of the histidine residues are affected by coenzyme binding. The largest effect-a marked upfield shift (0.85 ppm) of the C2 proton resonance-is seen for His-64. The hypoxanthine analogue of the coenzyme was found to produce a smaller upfield shift and, in addition, a decrease in the pK of His-64. The effects on this reductase are discussed in the light of the crystal structure [Matthews, D. A., Alden, R. A., Bolin, J. T., Filman, D. J., Freer, S. T., Hamlin, R., Hol, W. G. J., Kisliuk, R. L., Pastore, E. J., Plante, L. T., Xuong, N., & Kraut, J. (1978) J. Biol. Chem. 253, 6946], and it is concluded that His-64 is close to a carboxyl group in the free enzyme and that the hypoxanthine ring binds in a somewhat different orientation to the adenine ring. The effects on histidine resonances A, E, and G are significantly different for oxidized and reduced coenzymes. The changes in pK of the histidines giving rise to resonances A and E (probably His-22 and His-18) are discussed in terms of ligand-induced conformational changes, which differ for NADP+ and NADPH.

Proton nuclear magnetic resonance study on the roles of histidine residues in the binding of polypeptide chain elongation factor Tu from Thermus thermophilus with aminoacyl transfer ribonucleic acid and guanine nucleotides.

Proton nuclear magnetic resonance (1H NMR) spectra were measured of the polypeptide chain elongation factor Tu (EF-Tu) from an extreme thermophile, Thermus thermophilus HB8 [Nakano, A., Miyazawa, T., Nakamura, S., & Kaziro, Y. (1979) Arch. Biochem. Biophys. 196, 233-238], in order to elucidate the environment around functionally important histidine residues. In the present study, the behavior of five histidine C2 proton signals was studied in more detail. A hydrogen-deuterium exchange experiment was carried out on the histidine C2 protons of free EF-Tu, and the previous assignments of C2 proton signals were revised in part. An analysis of the 1H NMR spectra of EF-Tu photooxidized under various conditions indicates that a histidine residue is located in the aminoacyl-tRNA binding site and is probably essential for the binding with aminoacyl-tRNA. A solvent-accessible histidine residue is found to lie near the aminoacyl-tRNA binding site. Furthermore, the effect of paramagnetic hexacyanochromate(III) ion on the 1H NMR spectra of free EF-Tu suggests that another histidine residue lies near the guanine nucleotide binding site.

Conformation of ribonuclease S-protein.

Ribonuclease S-protein exhibits a pH-dependent conformational transition between folded and unfolded states, and some unfolded S-protein persists up to pH 8. The histidine C2 proton resonance of the unfolded species was erroneously assigned by Bradbury et al. to histidine residue 119 of the folded species.

High resolution proton nuclear magnetic resonance studies of interaction between deoxyhaemoglobin and small molecules. Dithionite and diphosphoglycerate

90 MHz proton n.m.r. spectra have been measured on solutions of deoxyhaemoglobin (Hb) in D2O in the presence and absence of dithionite (DT) and D-2,3-diphosphoglycerate (DPG) ions.Application of deconvolution and interpolative baseline correction software has enabled the pH dependence of the histidine C2 proton chemical shifts to be observed without the necessity of using high fields or elaborate pulse sequences. The n.m.r. data are interpreted in terms of a strong fast exchange interaction with DPG and a much weaker one, at the same site, for DT.Histidines β2 and β143 at the DPG binding site have been assigned to peaks in the proton n.m.r. spectrum. From the chemical shift behaviour of a selected resonance at different DPG concentrations, it has been shown that DPG and Hb bind with a 1:1 stoichiometry. The measured affinity constant is (5.1 ± 0.4)× 104 mol–1 and is independent of the ionised species of DPG present in the pH range 7.1–7.3.

1H Nuclear magnetic resonance spectroscopy of yeast copper-zinc superoxide dismutase. Structural homology with the bovine enzyme

High resolution nuclear magnetic resonance (n.m.r.) spectroscopy of the copper-zinc superoxide dismutase from Saccharomyces cerevisiae has revealed a substantial structural homology with the bovine enzyme. N.m.r. spectra of the apo enzyme and the holo-reduced and holo-oxidized enzymes are reported and assignments are made to the histidines in the active site and the single tyrosine residue. All the assignments are in agreement with the known amino-acid sequence and the geometry of the active site is virtually unchanged. Addition of halide ions to the reduced holo enzyme results in the perturbation of the chemical shift of three histidine C2 protons and the degree of perturbation is Cl−∼Br−>I−>F− for 1m solutions of the anions. The enzyme has also been shown to retain its structure at 75°C.

Nuclear magnetic resonance titration curves of histidine ring protons. Human metmyoglobin and the effects of azide on human, horse, and sperm whale metmyoglobins.

Four titrating histidine ring C2 and C4 proton resonances are observed in 220 MHz proton NMR spectra of human metmyoglobin as a function of pH. Values of ionization constants determined from the NMR titration data using an equation describing a simple proton association-dissociation equilibrium are curves (1) 6.6, (2) 7.0, (3) 5.8, and (4) 7.4. Four histidine residues have also been found to be solvent-accessible in human metmyoglobin by carboxymethylation studies (Harris, C.M., and Hill, R.L. (1969) J. Biol. Chem. 244, 2195-2203). Two of the titration curves (3 and 4) deviate significantly from the chemical shift values normally observed for histidine C2 proton resonances. Curve 3, with a low pKa, is shifted downfield at high values of pH and also exhibits a second minor inflection with a pKa value of 8.8. On the other hand, the high pKa curve, 4, is shifted upfield at all values of pH. The characteristics of the NMR titration curves with the lowest and highest pKa values (3 and4) are very similar to curves observed previously with sperm whale and horse metmyoglobins (Cohen, J.S., Hagenmaier, H., Pollard, H., and Schechter, A.N. (1972) J. Mol. Biol. 71, 513-519). These results indicate that the histidine residues from which these curves are derived have unusual and characteristic environments in this series of homologous proteins. The NMR spectra of all three metmyoglobins are changed extensively as a result of azide ion binding, indicating conformational changes affecting the environments of several imidazole side chains. The presence of azide ion causes a selective downfield chemical shift for the low pKa curve and a selective upfield chemical shift for the high pKa curve in all three proteins. Azide also abolishes the second inflection seen in the low pKa curve at high pH. In addition to these effects, the presence of azide ion permits the observation of two additional titrating proton resonances for all three metmyoglobins. Increasing the azide to protein ratio at several fixed values of pH yields results which show that a slow exchange process is occurring with each of the metmyoglobins. In the azide titration studies the maximum changes in the NMR spectra occurred at approximately equimolar concentrations. The NMR results for these proteins in the absence and presence of azide ion are related to x-ray crystallographic studies of sperm whale metmyoglobin and the known alkylation properties of the histidine residues. Tentative assignments of the titrating resonances observed are suggested.

Correlation of exchangeable (NH) and nonexchangeable (C2H) histidine resonances in the proton nmr spectrum of ribonuclease A in aqueous solution

AbstractThe ionization characteristics of the hydrogen‐bonded His 12 N1 proton observed to titrate between 11 to 13 ppm in the nmr spectrum of ribonuclease A in H2O solution are compared with the ionization characteristics of the four histidine C2 protons in the enzyme. Comparison of the pKa's of the enzyme in H2O and D2O in the absence and presence of cytidine monophosphate (−5′, −3′, and −2′) inhibitors, line widths in the presence of Cu II at pH 3.6 and 5.6, and chemical shifts in the presence of AgNO3 permit a correlation of the exchangeable His 12 N1 proton with the active site histidine C2 proton exhibiting the lower ionization pKa. The histidines with pKa of 5.1 and 5.6 in ribonuclease A in the absence of salt are assigned in this study to His 12 and His 119, respectively.

Nuclear magnetic resonance spectroscopy of denatured proteins

The proton magnetic resonance spectroscopy of 11 proteins (molecular weight range 5700-650000) has been investigated in five denaturing solvents, viz., trifluoroacetic acid-d, formic acid, dichloroacetic acid, 6M guanidine hydrochloride in D2O, and 8M urea in D2O. The chemical shifts, line-widths, and intensities of the resonances have been measured of the histidine C2 protons, the methionine SCH3 protons and methyl protons of leucine, isoleucine, and valine, the aromatic protons, and the α-CH protons. ��� It is found that, with some exceptions delineated below, the line- widths of the methyl resonances are constant for a particular solvent, independent of the molecular weight of the protein. This indicates that, in general, the proteins behave as random coil structures in these solvents, which confirms the conclusion reached by Tanford and co-workers1-4 for 6M guanidine hydrochloride. ��� However, methyl line broadening occurs in dichloroacetic acid for catalase and fibrinogen, in guanidine hydrochloride for insulin, and in urea for insulin and lysozyme. Furthermore, the C 2 histidine resonance is absent in dichloroacetic acid solutions of thyroglobulin, catalase, and fibrinogen; the SCH3 resonance is absent in myoglobin in trifluoroacetic acid-d and occurs as a doublet for trypsin in guanidine hydrochloride and in urea. A general line broadening of resonances indicates association and/or incomplete unfolding of molecules, whereas perturbations of only one particular resonance, as in the cases detailed above, are probably due to intramolecular non-covalent interactions which involve the perturbed group and another unspecified group in the protein. ��