Interaction of tropomyosin and troponin T: a proton nuclear magnetic resonance study

Proton nuclear magnetic resonance (1H NMR) has been used to study the nature of the interaction between tropomyosin (TM) and troponin T (Tn-T). Resonances corresponding to the histidine residues in fragments of both TM and Tn-T can be resolved and assigned in the 1H NMR spectrum. Changes in the pH titration profiles of these resonances when the various fragments are mixed provide probes of the interaction sites between the proteins. Fragment T1 (residues 1-158) of Tn-T appears to interact weakly but specifically with fragments of TM in which the NH2-terminus (residues 1-10) is intact. While fragment CB2 (residues 71-151) of Tn-T interacts weakly (dissociation constant of 0.1-0.2 mM) with NH2-terminal fragments of TM, this appears to be nonspecific since the absence of residues 1-10 and 128-189 of TM does not affect the observed perturbations of the titration profiles of His-79 of CB2. Although a strong interaction between T1 and the COOH-terminal Cy2 fragment (residues 190-284) of TM has been previously demonstrated, no perturbation of His-276 of Cy2 or of His-7, -23, -29, or -36 of T1 was observed in a mixture of T1/Cy2. The pKa of His-276 was also not affected in a mixture of Cy1/Cy2 (where Cy1 is residues 1-189 of TM) but was significantly decreased in the ternary complex T1/Cy1/Cy2. The importance of residues 1-70 of Tn-T in its binding to TM is illustrated by the specificity it confers on the T1/Cy1 interaction and by the absence of His-276 perturbation in the mixture CB2/Cy1/Cy2.     

Interaction of human adult methemoglobin in low-spin state with inositol hexaphosphate. A proton magnetic resonance study

The hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of the low-spin complexes of human adult methemoglobin were found to be much altered by the addition of inositol hexaphosphate (IHP). The stoichiometry and pH-dependence of IHP binding, and the spin equilibrium of azide methemoglobin are parallel to those of high-spin human methemoglobin and of carp methemoglobin, both of which are proposed to be switched from the R to T states with IHP. The present NMR results show that IHP affects the structure of human methemoglobin regardless of the spin state of the heme iron, suggesting that there is no correspondence between quaternary structure and the spin state of ferric heme iron.     

A proton nuclear magnetic resonance study of sulfmyoglobin cyanide

The proton nuclear magnetic resonance spectrum of sulfmyoglobin cyanide was studied at 400 MHz. The position of a methyl-group resonance at low field is consistent with a chlorin-like structure for the prosthetic group. The proton NMR spectrum of the cyanide derivative of the purified prosthetic group which decomposes upon extraction from the protein was found to be the same as that of the cyanide derivative of the prosthetic group extracted from myoglobin and a sample prepared from hemin-Cl.     

Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study

The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.     

A proton magnetic resonance study of water in human stratum corneum

Proton magnetic resonance spectroscopy has been used to study the nature of water in human stratum corneum. For a single planar sheet of stratum corneum mounted at a specific orientation to the applied magnetic field, three distinct absorptions may be seen having different chemical shifts and spin-lattice relaxation times (T₁). All T₁ values for these resonances are smaller than that for normal liquid water. One absorption is unusual in that the resonance position is dependent upon the orientation of the sample within the field.     

A proton nuclear magnetic resonance study of the interaction of cadmium with human erythrocytes

The binding of Cd²⁺ by molecules in the intracellular region of human erythrocytes has been studied by ¹H-NMR spectroscopy. From changes in spin-echo Fourier transform NMR spectra for both intact and hemolyzed erythrocytes to which CdCl₂ was added, direct evidence was obtained for the binding of Cd²⁺ by intracellular glutathione and hemoglobin. Time-courses were measured by ¹H-NMR for the uptake of Cd²⁺ by intact erythrocytes in saline/glucose solution and in whole blood. In both cases, the uptake, as indicated by changes in the ¹H-NMR spectrum for intracellular glutathione, plateaus after about 30 min. The effectiveness of the disodium salt of EDTA and of various thiol-chelating agents for releasing glutathione from its Cd²⁺ complexes in hemolyzed erythrocytes was also studied. EDTA was found to be more effective than thiols, and dithiols more effective than monothiols.     

Calcium binding by troponin-C. A proton magnetic resonance study.

The conformational changes induced by the binding of Ca(II) to rabbit skeletal muscle troponin-C (TNC) have been followed by proton magnetic resonance spectroscopy. Ca(II)-free TNC (apo-TNC) contains definite ordered regions. Ca(II) titration of the high affinity sites (cf. Potter , Gergely, 1975) causes a large folding of the backbone, some of which involves refolding of an ordered region(s) and changes in several side-chains e.g. Glu, Asp and Phe. Titration of the low affinity sites does not alter the backbone but leads to changes among hydrophobic side-chains (one or more Val, Leu, Ile; two or more Phe, Glu and Asp) that define an ordered region(s) of apo-TNC. The rate constants for the conformation changes of the low and high affinity sites are approximately 10 s^?1 and < 20 s^?1, respectively. Final stages of the titration include a downfield shifted methyl group (likely Ile) and a Phe residue. The thermal stabilities of apo-TNC, TNC · Ca₂(II) and native TNC were compared. It was concluded that Ca(II) binding by the two high affinity sites both directs and stabilizes much of the structure. The role of the changes of the low affinity sites, which are thought to activate contraction, are briefly discussed.     

Conformation of malformin A: a proton magnetic resonance and a circular dichroism study

Malformin A is a cyclic pentapeptide with an intramolecular disulfide bridge. The conformation in solution of this molecule has been studied by NMR and CD. The 270 MHz Proton spectrum in dimethyl sulfoxide is well resolved and the peaks corresponding to the five residues have been assigned. From the temperature dependence of chemical shifts of the peptide protons and from the exchange rate of these protons, it is concluded that the NH proton of one Cys is shielded from the solvent. This observation and H? N? _αC? H angles, estimated from the corresponding coupling constants, a proposed conformation of the peptide backbone. From the H? _βC? _αC? H coupling constants, a P chirality for the disulfide bridge is proposed. Such a conformation is confirmed by the circular dichroism spectrum which shows a negative band at λ > 250 nm. It is concluded that the conformation of malformin A is rigid and that the disulfide bridge is exposed to interact with biological receptors.     

Halide interaction with phospholipids: proton magnetic resonance studies

Water dispersions of egg phosphatidyl choline, dioleoyl phosphatidyl choline and lyso egg phosphatidyl choline have been studied by means of 220 MHz proton magnetic resonance techniques. The N⁺(CH₃)₃ proton signal, for phosphatidyl choline vesicles, consists of two components. The two components are thought to arise from N⁺(CH₃)₃ groups interior and exterior to the phosphatidyl choline vesicle. Anions were found to increase the separation of the two components. The effectiveness of the anions follows their order in the lyotropic series. For a given anion, the increase in component separation depends on the nature of the phospholipid. Iodine was found to modify the anion effect. The results are related to the results of other workers on water transport across lipid bilayer membranes and on phospholipid-halide-binding.     

A proton nuclear magnetic resonance study on the solution structure of crotamine

Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. ThepH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidicpH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for He-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.     

A proton nuclear magnetic resonance study of the interaction of mercury with intact human erythrocytes

The binding of mercuric ion (Hg(II)) by small molecules in the intracellular region of intact human erythrocytes has been studied by ¹H-NMR spectroscopy. HgCl₂ added to intact erythrocytes in saline-glucose suspension is found to cross the membrane and reach an equilibrium distribution among the molecules of the erythrocyte within 4 min. In the intracellular region Hg(II) reacts with GSH and hemoglobin to form the ternary mixed-ligand complex GSH-Hg(II)-hemoglobin. The analogous complex with ergothioneine is formed after all the GSH is complexed. ¹H-NMR spectra show that the GSH-Hg(II)-hemoglobin complex also forms in simpler solutions containing HgCl₂, GSH and hemoglobin, whereas the complex Hg(GSH)₂ predominates in solutions of GSH and HgCl₂. The lifetime of the GSH in the GSH-Hg(II)-hemoglobin complex is shown to be less than 30 s, which provides direct evidence for the first time that Hg(II) complexes in biological systems are quite labile, even though their thermodynamic stability is large. The effectiveness of eight sulfhydryl-containing ligands, some of which have been used as antidotes for Hg(II) poisoning, for releasing GSH from its Hg(II) complex in hemolyzed erythrocytes was also studied. Dithiol ligands were found to be more effective than monothiols, with dithioerythritol the most effective of the dithiols.     

Fusion kinetics of phosphatidylcholine-phosphatidic acid mixed lipid vesicles: A proton nuclear magnetic resonance study

The kinetics of Ca²⁺-induced fusion of phosphatidylcholine-phosphatidic acid vesicles has been studied using the dependence of proton nuclear magnetic resonance linewidths on vesicle size. The linewidth of the lipid acyl chain methylene resonance been shown to be sensitive to changes in vesicle size but insensitive to vesicle aggregation. For vesicle systems with the same lipid composition, the linewidth increases in a linear fashion with vesicle radius over the range 125–300 Å. This dependence has been used to determine quantitatively fusion rates and the dependence of such rates on Ca²⁺ as well as an vesicle concentration. For vesicle concentrations in the range of 3 · 10^?6–10^?5 M and Ca²⁺ concentration at a level approaching 1 : 1 with respect to phosphatidic acid, the initial fusion rates have been found to be fast, with half-times of 1–10 min. An order of reaction of 2.7 with respect to vesicle concentration has been observed. Mechanisms of vesicle fusion are discussed in view of these observations.     

A proton nuclear magnetic resonance study of gamma-glutamyl-amino acid cyclotransferase in human erythrocytes

Spin-echo NMR spectroscopy was shown to be a reliable technique for the monitoring of the in situ cleavage of gamma-Glu-Ala by gamma-glutamyl-amino acid cyclotransferase in whole erythrocytes and hemolysates. Of particular importance was the difference in chemical shifts between peptide resonances and those of the constituent amino acids. Using lysates of varying dilution, it was shown that the specific activity of the enzyme was not concentration-dependent, thus suggesting a lack of cytosolic low-molecular-weight-effectors or enzyme dissociation. Furthermore, the initial velocities of the reaction as a function of substrate concentration obeyed Michaelis-Menten kinetics with a Km = 2.0 +/- 0.3 mmol/l and Vmax = 137 +/- 7 mmol/h/l of cell water in 1H2O medium. Similar analysis in 2H2O medium revealed a solvent kinetic isotope effect of 1.9 +/- 0.4 at low substrate concentrations. The implications of this observation for the mechanism of the reaction are discussed. Cleavage of the peptide by a suspension of intact erythrocytes was at a rate 300 times less than the corresponding lysate flux, thus indicating the rate limitation by transport in the coupled system.     

Assignment, by proton magnetic resonance, of histidines of dihydrofolate reductase from chicken liver

The effects of pH and temperature upon C epsilon 1 H resonances of the four histidyl residues of chicken liver dihydrofolate reductase in binary complex with methotrexate were studied by 500-MHz 1H NMR spectroscopy. The four histidines labelled a, b, c, d are distinguishable by their pK values and the chemical shifts of their C epsilon 1H protons. The local electromagnetic environment as deduced from X-ray studies at 2.9 A resolution was used as a basis for proposed assignment of the four histidines. The assignments were a: H42, b: H140, c: H131, d: H87. Furthermore the histidyl residue labelled c was shown to be upfield shifted in its C epsilon 1H proton in the enzyme-methotrexate complex compared to the native enzyme. The hypothesis of a conformational change of the protein is discussed.