Dimerization of native and proteolytically modified neurophysins as monitored by proton magnetic resonance spectroscopy: proximity of tyrosine-49 to the subunit interface

Neurophysin is a self-associating protein in which peptide-hormone binding and dimerization are thermodynamically linked. The structural basis of the linkage is unknown. We have studied the dimerization of bovine neurophysin I and two proteolytically modified derivatives by proton nuclear magnetic resonance spectroscopy in order to identify residues at the intersubunit contact regions and to evaluate the origin of the reported loss of dimerization associated with tryptic excision of residues 1-8. The concentration dependence at neural pH of the spectra of native neurophysin and des-90-92-neurophysin demonstrated a finite set of dimerization-sensitive resonances that included the ring protons of Tyr-49. Using these to monitor dimerization, we confirmed predictions of a large increase in the dimerization constant associated with carboxyl protonation. By the same criteria, dimerization of the des-1-8 protein, in disagreement with earlier reports, was found to be undiminished relative to that of the native protein. However, spectral changes in the Tyr-49 ring ortho proton region associated with dimerization of the des-1-8 protein differed significantly from those in the native protein and indicated an altered conformation of the des-1-8 dimer apparently restricted to the vicinity of Tyr-49. The results are shown to place Tyr-49 adjacent to both the intersubunit contact region and the 1-8 sequence in the native protein, loss of stabilizing interactions with 1-8 leading to altered interactions of Tyr-49 with the subunit interface. Because Tyr-49 is also close to the peptide-binding site, this arrangement spatially links the peptide-binding and dimerization sites of neurophysin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance studies on the iodide binding by horseradish peroxidase

Binding of an iodide ion to horseradish peroxidase was studied by following the hyperfine-shifted proton nuclear magnetic resonance signals of the enzyme. For the enzyme in an iodide-free solution, the spectra of hyperfine-shifted methyl region were only slightly affected by varying pH. In the presence of iodide (200 mM), however, both chemical shifts and line widths of the heme peripheral 1- and 8-methyl proton signals were markedly affected by the pH change from 7 to 4 and broadened at pH 4. From the change in peak heights of these signals at various concentrations of iodide, the dissociation constant of the iodide to the enzyme was calculated to be about 100 mM at pH 4.0. The peak derived from the proximal histidyl imidazole N epsilon-H proton was not perturbed by the addition of 200 mM iodide at pH 4.0 and 7.1. The rate of oxidation of iodide with hydrogen peroxide catalyzed by the enzyme was increased with decreasing pH, indicating the participation of an ionizable group with the pKa value of 4.0. Optical difference spectrum studies showed that iodide exerts no effect both at pH 4.0 and 7.4 on the binding affinity of resorcinol which is associated with the enzyme in the vicinity of the heme peripheral 8-CH3 group. These results suggest that an iodide ion binds to the enzyme at almost equal distance from the heme peripheral 1- and 8-methyl groups at the distal side of the heme and that the interaction becomes stronger in acidic medium with protonation of the ionizable group with the pKa value of 4.0.     

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹ and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, β-branched D -aliphatic residues at position 7 combined with bulky L -aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D -Arg⁰, Hyp³, Thi⁵, D -Cpg⁷, Cpg⁸]-BK [Cpg: α-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L -proline; Thi: β-(2-thienyl)-L -alanine] ( I ), [D -Arg⁰, Hyp³, D -Cpg⁷, Cpg⁸] -BK ( II ), as well as its variant with D -Cpg⁷ replaced by Cpg⁷, namely [D -Arg⁰, Hyp³, Cpg⁷, Cpg⁸]-BK ( III ). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II , in direct correlation with the peptide activities. No turn-like structure was found for residues 6–9. In peptide III , a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists ( I , II ) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D -Cpg⁷]-BK ( IV ) showed no defined secondary structure. © 1993 John Wiley & Sons, Inc.     

Proton magnetic resonance studies of alpha-keto acids.

Pulsed Fourier transform proton magnetic resonance was used to study alpha-ketoglutaramic, and several other alpha-keto acids in aqueous solutions as a function of pH. Most alpha-keto acids were found to exist in equilibrium with the hydrate (gem-doil). The equilibrium position favors the nonhydrated alphs-keto acid at neutral pH, but at low pH values (below the pKa of the alpha-carboxylic acid group) the hydrate predominates. We found evidence that alpha-ketoglutaric acid exists in a third equilibrium form which is assigned to the lactol. alpha-Ketoglutaramic acid (the alpha-keto acid analog of glutamine) which is known to exist predominantly in a cyclic form at pH 7.0 was shown to exist as a cyclic structure over a wide pH range. However, the cyclic form is an equilibrium mixture of 2-pyrrolidone-5-hydroxy-5-carboxylic and 1-pyrrolin-2-one-5-carboxylic acids.     

Proton and iodine-127 nuclear magnetic resonance studies on the binding of iodide by lactoperoxidase

Interaction of an iodide ion with lactoperoxidase was studied by the use of 1H NMR, 127I NMR, and optical difference spectrum techniques. 1H NMR spectra demonstrated that a major broad hyperfine-shifted signal at about 60 ppm, which is ascribed to the heme peripheral methyl protons, was shifted toward high field by adding KI, indicating the binding of iodide to the active site of the enzyme; the dissociation constant was estimated to be 38 mM at pH 6.1. The binding was further detected by 127I NMR, showing no competition with cyanide. Both 1H NMR and 127I NMR revealed that the binding of iodide to the enzyme is facilitated by the protonation of an ionizable group with a pKa value of 6.0-6.8, which is presumably the distal histidyl residue. Optical difference spectra showed that the binding of an aromatic donor molecule to the enzyme is slightly but distinctly affected by adding KI. On the basis of these results, it was suggested that an iodide ion binds to lactoperoxidase outside the heme crevice but at the position close enough to interact with the distal histidyl residue which possibly mediates electron transport in the iodide oxidation reaction.     

Proton magnetic resonance studies of the states of ionization of histidines in native and modified subtilisins

A technique was developed to exchange the backbone -N-H protons in D2O in the native subtilisins Carlsberg and BPN (Novo) that resulted in clearly resolved proton resonances in the aromatic region of the nuclear magnetic resonance spectrum. pH titration curves for four of the five histidine C2-H resonances in subtilisin Carlsberg and five of the six in subtilisin BPN between 7.5 and 8.8 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid sodium salt provided microscopic pKa's between 6.3 and 7.2 for both sources of the enzyme at ambient (approximately 22 degrees C) probe temperature. A resonance that titrated with a pKapp of 7.35 +/- 0.05 was observed in the 1H spectra only of the diisopropylphosphoryl derivatives of the subtilisins from both sources. The 31P NMR pH titration of the same preparations under identical conditions of solvent (D2O) and temperature gave a pKapp = 7.40 +/- 0.05 of the single titratable resonance. Both observations must pertain to His-64 at the active center. A resonance smaller than the others and titrating with a pKapp of 7.2 could also be observed in the native enzymes. This resonance was assigned to the catalytic center histidine since its pK corresponded to that derived from kinetic studies. No major perturbations in the chemical shifts or the pK's derived from the pH dependence of the observed resonances were apparent in the presence of saturating concentrations of the two putative transition-state analogues phenylboronic acid and bis [3,5-(trifluoromethyl)phenyl]boronic acid and in monoisopropylphosphorylsubtilisin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.     

Proton magnetic resonance studies on dermorphin and its peptide fragments

Dermorphin (Tyr? D-Ala? Phe? Gly? Tyr? Pro? Ser? NH₂), a potent natural peptide opioid, its synthetic L-Ala² analog, and all the N fragments from the tripeptide (Tyr? D -Ala? Phe? NH₂) to the parent hexapeptide amide were characterized for the first time by means of proton nmr spectroscopy at 11.74 T. Assignments of most protons of dermorphin were facilitated by the study of the N-terminal fragments. Comparison of spectroscopic parameters with relative pharmacological activity is proposed as a possible means of studying flexible agonists in solution.     

Proton nuclear magnetic resonance studies on the kinetics of tryptic fragments of calmodulin upon calcium binding

The kinetics of the Ca2+-dependent conformational change of the tryptic fragments F12 (residues 1-75) and F34 (residues 78-148) of calmodulin were studied by 1H-NMR. Resonances of two phenylalanines, 16 (or 19) and 65 (or 68), N epsilon, N epsilon, N epsilon-trimethyllysine-115 and tyrosine-138 were examined by the saturation-transfer technique or computer-aided line-shape simulation to obtain the rate of the conformational exchange between the Ca2+-free form and the Ca2+-bound form. The rates for F12 and F34 in the presence of 0.2 M KCl at 22 degrees C were 300-500 s-1 and 3-10 s-1, respectively. Activation parameters are as follows: Delta H not equal to = 11(+/- 2) kcal X M-1 and delta S not equal to = -9(+/- 5) cal X K-1 X M-1 for F12, and delta H not equal to = 16(+/- 2) kcal X M-1 and delta S not equal to = -2(+/- 5) cal X K-1 X M-1 for F34. These kinetic data for the conformational exchange are in agreement with those of Ca2+ dissociation from the binding sites obtained by 43Ca-NMR and stopped-flow fluorescence studies.     

Proton magnetic resonance studies of peroxidases from turnip and horseradish.

Proton NMR spectra at 270 MHz have been measured for horseradish peroxidase and turnip peroxidase isoenzymes (P1, P2, P3 and P7) in both their high spin ferric native states and as the low spin ferric cyanide complexes. Resonances of amino acids near the heme have been identified and used to investigate variations in the structure of the heme crevice amongst the enzymes. Ligand proton resonances have been resolved in spectra of the cyanide complexes of the peroxidases and these provide information on the heme electronic structure. The electronic structure of the heme and the tertiary structure of the heme crevice are essentially the same in the acidic turnip isoenzymes, P1, P2 and, to a lesser extent, P3 but differ in the basic turnip enzyme, P7. The heme electronic structure and nature of the iron ligands in peroxidases are discussed. Further evidence is presented for histidine as the proximal ligand. A heme-linked ionizable group with a pK of 6.5 has been detected by NMR in the cyanide complex of horseradish peroxidase.     

Proton nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome.

Rhodospirillum rubrum cytochrome c2 was studied by proton nuclear magnetic resonance at 220 MHz. Assignments were made to the resonances of heme c by double-resonance techniques and by temperature-dependence studies. The aromatic resonances of Trp-62 and Tyr-70 of ferrocytochrome c2 were identified by spin-decoupling experiments. The resonances of the Met-91 methyl group of the ferri- and ferrocytochromes were assigned by saturation-transfer experiments. The assignments are compared to those made for cytochromes c. A pH titration showed that the methionine methyl resonance of ferricytochrome c2 shifted with a pK of 6.25 and disappeared above pH 9. No histidine CH resonances that titrated normally over the neutral pH range were observed in the spectrum of either oxidation state of the protein. The possible origins of the ionizations at pH 6.25 and 9 are discussed.     

Proton magnetic resonance studies of human adult hemoglobin in water

The preferential binding of oxygen to the α hemes of normal human adult hemoglobin in the presence of organic phosphates in D₂O at neutral pD has been confirmed in H₂O. Additionally, it has been found that the preferential binding of oxygen is pH dependent with random ligation being observed at pH ~ 9.     

Proton magnetic resonance spectroscopic studies of proteins containing deuterated tryptophan residues

Summary The deuteration of the tryptophan residues of hen egg white lysozyme, bovine-lactalbumin and bovine-lactoglobulin in d-TFA has been studied by PMR spectroscopy. It is found that short times of exposure to d-TFA allow selective deuteration at the C-2 position with only a small amount of deuteration at the C-5 position, as expected from studies on model peptides described in the previous paper. The proteins studied essentially regained their native structures after the treatment, except for broadening and shifting of the histidine resonances in the case of-lactalbumin. Selective deuteration at the tryptophan C-2 position was readily observed by difference spectroscopy of the denatured protein, but PMR difference spectra of the same proteins in benign solvents did not contain resonances from all of the exchanged protons. Some resonances could not be observed because of line broadening, which causes the resonances to fall below the limit of sensitivity of detection at 100 MHz. Deuteration by brief exposure to d-TFA should be useful for the identification of tryptophan resonances in the PMR spectra of native proteins.The deuteration of all the aromatic protons of tryptophan residues in proteins by immersion in d-TFA for 4 hours at room temperature was studied. This technique is unlikely to be of general use for the simplification of the aromatic region of the PMR spectra of native proteins because of the degradation of tryptophan residues which results from the acid treatment.An invited article.     

Proton nuclear magnetic resonance studies of intact native bovine parathyroid hormone

Native intact bovine PTH was studied by proton nuclear magnetic resonance (NMR) techniques, at pH 3.5 and pH 6.3. The 1H-NMR spectra had good resolution and many multiplet structures were observed. Assignment of the NMR resonances corresponding to specific amino acids was approached using 1H chemical shifts, coupling constants, and pH dependence in the one-dimensional spectra and the 1H-1H connectivities revealed in two-dimensional homonuclear correlated spectroscopy (COSY) experiments. All the aromatic proton resonances were assigned. Two histidine residues had lower pK than the other two. The methyl groups of two residues were moved significantly downfield: using COSY and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) correlations, these were assigned to an alanine residue close to both Trp-23 and Tyr-43, and a valine residue in close spatial proximity to Trp-23. The NOESY spectrum also showed cross-peaks between the residues of the upfield valine-leucine-isoleucine methyl envelope. Many of the H alpha protons moved upfield as the pH was increased. These results indicate that intact native PTH exists in a preferred conformation in solution at pH 6.5. Our studies have provided new information on the three-dimensional spatial proximity of several amino acids along the polypeptide chain. The observed interactions are consistent with the currently accepted model suggesting that the hormone has two separate structural domains associated with the amino- and carboxy-terminal regions of the molecule respectively. The potential implications of this model for the expression of biological activity are discussed.