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H R Kalbitzer

Publications and source records attributed to H R Kalbitzer.

87 records · Page 5Linked to original sources
PubMed1984

Computer aided evaluation of two-dimensional NMR spectra of proteins.

A computer program for the automatic evaluation of two-dimensional NMR spectra of peptides and proteins has been developed. The used strategy is described, the advantages and limits of this approach are discussed. The program was successfully tested on a COSY-spectrum of the neuropeptide Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe from hydra, resulting in a drastic reduction of the time needed for the evaluation of two-dimensional NMR data.

Amino Acid Sequence↗
PubMed1984

Phosphoenolpyruvate-dependent phosphorylation site in enzyme IIIglc of the Escherichia coli phosphotransferase system.

Enzyme-IIIglc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphoenolpyruvate in a reaction requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIglc from E. coli and the characterization of the active center. Alkaline hydrolysis of [32P]P-IIIglc and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIglc of S. typhimurium. Here we present 1H-NMR measurements of IIIglc and P-IIIglc from E. coli which further substantiate that the phosphoryl group in P-IIIglc is linked to the N-3 position of a histidyl residue. After phosphorylation of IIIglc with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIglc on dodecylsulfate/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to unphosphorylated protein may indicate a structural change following phosphoenolpyruvate-dependent phosphorylation.

Binding Sites↗
PubMed1984

Electron-paramagnetic-resonance studies of manganese(II) complexes with elongation factor Tu from Bacillus stearothermophilus. Observation of a GTP hydrolysis intermediate state complex.

Changes in the coordination of Mn2+ to nucleotide, water and protein at the active site of elongation factor Tu (EF-Tu) have been studied by electron paramagnetic resonance (EPR) spectroscopy. From the time dependence of the Mn2+ spectrum after addition of GTP to EF-Tu X Mn, it was apparent that three complexes with different EPR linewidths could be detected. Using additional information from the kinetics of 32Pi production and release from EF-Tu X Mn X [gamma-32P]GTP these were identified as EF-Tu X Mn X GTP (linewidth 4.2 mT), EF-Tu X Mn X GDP X Pi (1.20 mT) and EF-Tu X Mn X GDP (1.29 mT). The linewidth for EF-Tu X Mn was 1.51 mT. The rate constant for GTP cleavage on EF-Tu was 0.01 min-1 at 24 C, for Pi release from the EF-Tu X GDP X Pi complex 0.0033 min-1. The corresponding rate constants in the presence of Mg2+ were 0.003 min-1 and 0.0065 min-1. The rate constant for reversal of the cleavage step was found to be much smaller than that for the rate of Pi release (and consequently much smaller than in the forward direction), as shown by 31P-NMR experiments on the incorporation of 18O into Pi from GTP hydrolyzed in the presence of H2 18O. EPR experiments using specifically 17O-labelled GTPs demonstrated an interaction of Mn2+ with the beta-phosphate in both the EF-Tu X GDP X Pi and EF-Tu X GDP complexes. Inorganic phosphate in the EF-Tu X GDP X Pi complex was found not to interact with the metal ion. From EPR experiments in H2 17O, it was concluded that the most probable number of water molecules in the different complexes was 4 (EF-Tu X Mn), 5 (EF-Tu X Mn X GDP X Pi) and 3 (EF-Tu X Mn X GDP), with 2, 0 and 2 metal-protein interactions respectively.

Binding Sites↗
PubMed1982

The structural isomerisation of human-muscle adenylate kinase as studied by 1H-nuclear magnetic resonance.

Human muscle adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3.) was studied by 1H-nuclear magnetic resonance spectroscopy. The C-2 and C-4 proton resonances of the active-center histidine His-36 could be identified; the pK of His-36 was determined as 6.1. The pK of His-189 is very low (4.9) although it is located at the surface of the protein. Other resonance lines are discussed in comparison with NMR spectra of porcine adenylate kinase [McDonald et al. (1975) J. Biol. Chem. 250, 6947-6954]. A pH-dependent structural isomerization as shown by X-ray crystallography in the pig enzyme [Pai et al. (1977) J. Mol. Biol. 114, 37-45] was not observed for human adenylate kinase in solution. However, the binding of adenosine(5')pentaphospho(5')adenosine (Ap5A), a bisubstrate inhibitor, to adenylate kinase causes an overall change of the NMR spectrum indicative of a large conformational change of the enzyme. The exchange rate (koff) for Ap5A was estimated as 10 s-1 and decreases by addition of Mg2+. On the basis of these values and the known dissociation constant it is likely that the binding of Ap5A is a diffusion-controlled process kon being 10(8) M-1 s-1. In conclusion, the system Ap5A/Mg2+/human adenylate kinase, which has been studied by NMR spectroscopy and X-ray diffraction in parallel, is suitable for analyzing the induced fit postulated by Jencks for all kinase-catalyzed reactions.

Adenylate Kinase↗
PubMed1982

HPr proteins of different microorganisms studied by hydrogen-1 high-resolution nuclear magnetic resonance: similarities of structures and mechanisms.

The HPr proteins of Streptococcus lactis, Streptococcus faecalis, Bacillus subtilis, and Escherichia coli were studied by 1H NMR at 360 MHz. The "active-center" histidines of all HPr proteins are characterized by a low pK value between 5.6 and 6.1 and similar spectral parameters. Phosphorylation of the histidyl residues leads to an increase of the pK value of 2-3 units and spectral changes characteristic for N-1 phosphorylation of the histidyl ring. The spectra of the HPr proteins of S. lactis, S. Faecalis, B. subtilis, and Staphylococcus aureus reveal many similarities, whereas the spectrum of the E. coli protein is different with exception of the active-center histidine. The HPr protein of S. lactis is formylated at its terminal amino group.

Bacillus subtilis↗
PubMed1982

The structure of the EF-Tu . GDP . Me2+ complex.

The structure of the MgGDP complex at the active site of elongation factor (EF-Tu) has been investigated by using phosphorothioate analogs of GDP in the absence and presence of various metal ions, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) measurements. The high stereoselectivity of EF-Tu for the diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDP[alpha S]) is independent of the nature of the metal ion and is caused by the interaction of the protein with the alpha-phosphate of GDP. By using GDP analogs where the oxygens at either the alpha-phosphate or the beta-phosphate have been selectively labelled with 17O and measuring their effect on the EPR spectrum of EF-Tu-bound manganese we are able to show that only the beta-phosphate of GDP is coordinated to the metal ion in the EF-Tu . Me2+ . GDP complex. 31P-NMR studies on GDP and guanosine 5'-O-(2-thiodiphosphate) (GDP[beta S]) bound to EF-Tu indicate that in the EF-Tu . Me2+ . GDP complex Mg2+ interacts more strongly with the beta-phosphate than with the alpha-phosphate. Together with binding studies using GDP[beta S] our NMR results also indicate that the protein is complexed to the beta-phosphorous of GDP via two oxygens.

Binding Sites↗
PubMed1981

Phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus: 1H nuclear magnetic resonance studies on phosphorylated and unphosphorylated factor IIIlac and its interaction with the phosphocarrier protein HPr.

The trimeric phosphocarrier protein factor III specific for galactosides was investigated by 1H NMR spectroscopy. The protomer contains four histidyl residues with acidic pK values in the range 5.6-6.2. One of the histidyl residues, His-B, carries the phosphoryl group. The pK value of His-B increases from 6.0 to 8.6 upon phosphorylation. To determine the position of the phosphoryl group with respect to the nitrogens required the isolation of a peptide T-2 containing the phosphorylated active-center histidine and one of the other histidines. The pK value and the chemical shift of the phosphopeptide clearly indicated the phosphorus to be bound to the N-3 atom of the imidazole ring. The temperature dependence of the factor III spectrum demonstrates multiple conformations which exchange rapidly on the NMR time scale. Titration of factor III with HPr protein showed an upfield shift of the active-center histidine, indicating complex formation between both proteins. Phosphorylation of both proteins abolished the interaction, which is plausible from mechanistic considerations.

Bacterial Proteins↗
PubMed1981

1H nuclear magnetic resonance studies on the structure and mechanism of the HPr protein of Staphylococcus aureus.

1H NMR studies of the phosphocarrier protein HPr and its three nitrotyrosyl derivatives revealed some structural features which may finally lead to an explanation of the mechanism of the phospho-transfer reaction. Titration studies on mononitrated, dinitrated, and trinitrated derivatives--i.e., derivatives with Tyr-56, Tyr-56 and Tyr-37, and Tyr-56, Tyr-37, and Tyr-6 modified--have been performed. The three tyrosyl residues seem to be in positions completely different from each other with respect to their solvent accessibility; Tyr-56 seems to be located near the surface of the protein, Tyr-6 seems to be completely buried, and Tyr-37 takes an intermediate position. Tyr-6 contributes to the core structure of the protein. A resonance at -0.18 ppm could be shown to correspond to a CH3 group of a valine. Nuclear Overhauser experiments revealed its being close to Tyr-6. One of the resonances tentatively assigned to methionine SCH3 groups titrates in the dinitrated derivative with the same pK as nitrotyrosyl residue 37. The titration behavior of the active-center histidyl residue suggests a hydrogen bond to the imidazole ring, possibly from Tyr-56 or Arg-17.

Bacterial Proteins↗
PubMed1981

The binding of nucleotides and metal ions to elongation factor Tu from Bacillus stearothermophilus as studied by equilibrium dialysis.

EF-Tu from B. stearothermophilus binds divalent metal ions even in the absence of guanine nucleotides. The association constants necessary for characterizing the multiple equilibria between EF-Tu, GDP and the divalent ions magnesium and manganese were determined by equilibrium dialysis. The constants are 4.6 X 10(4) M-1 and 5.4 X 10(5) M-1 for the binding of Mg2 and 1.0 X 10(5) M-1 and 1.1 X 10(6) M-1 for the binding of Mn2 to EF-Tu and EF-Tu . GDP, respectively. In the absence of divalent ions EF-Tu binds GMP, GDP and GTP with association constants of 3 x 10(3) M-1, 1.7 x 10(7) M-1 and 1.3 x 10(6) M-1, respectively. The binding of GDP in the presence of metal ions is an order of magnitude stronger than in the absence of metal ions.

Bacterial Proteins↗
PubMed1978

The binding of calcium and magnesium to sarcoplasmic reticulum vesicles as studied by manganese electron paramagnetic resonance.

The binding pattern of the biologically relevant ions calcium and magnesium has been investigated via the binding of the ion-analogue manganese. The binding parameters of manganese are obtained conveniently by standard electron paramagnetic resonance techniques, the binding of calcium and magnesium is inferred from competition experiments. The quantitative analysis was carried out with a computer program which was able to treat the competition of three kinds of ions for three classes of independent and one class of cooperative binding sites. It was possible to correlate specific binding classes with biological functions of the sarcoplasmic reticulum membrane. The magnesium and manganese specific binding class of medium affinity is related to the catalytic function of these ions in the ATP-splitting. For the first time a manganese and calcium specific class of cooperative binding sites has been observed and it can be related to the inhibition of the calcium transport at high concentrations of manganese or calcium. From the binding results a model can be developed which allows a full description of the role of the divalent ions and their specific binding sites during calcium transport.

Adenosine Triphosphatases↗
PubMedDate not supplied

A new high sensitivity 19F probe for labeling cysteine groups of proteins.

A novel iodoacetamide label 4-perfluoro-tert-butyl-phenyliodoacetamide (PFP) containing nine fluorine atoms in equivalent positions has been synthesized. It provides a homogeneous 19F NMR resonance line which can be detected with high sensitivity when coupled to proteins. As an example, the sulfhydryl groups of actin have been labeled with PFP; < 100 nmol of this medium sized protein (corresponding to 2.5 mL of a 40 microM solution) can be detected easily in a single scan at 470 MHz.

Acetamides↗
PubMedDate not supplied

Mechanism of the activation of proteinase inhibitor synthesis by systemin involves beta-sheet structure, a specific DNA-binding protein domain.

We analyzed a tertiary structure of systemin, the first identified polypeptide plant hormone, using two-dimensional NMR spectroscopy. From these data and molecular dynamics calculations we concluded that the peptide can adopt a Z-like-beta-sheet structure, which has previously been found in many specific DNA-binding proteins. Using DNA-cellulose affinity chromatography, we showed that systemin binds strongly to DNA. We suggest that the specific systemin-DNA interaction, particularly in a promoter region of the proteinase inhibitors, could effect gene expression and thus explain the biological activity of systemin.

Amino Acid Sequence↗
PubMedDate not supplied

On the analysis of competitive binding of various ligands to cooperative and independent binding sites of macromolecules.

The paper deals with the practical evaluation of multiple binding equilibria of macromolecules and different ligands competing for the same binding sites. The necessary formalism is reviewed and set up for the equilibria involving a macromolecule with various classes of independent binding sites and/or a class of cooperative sites and up to three different ligands in competition for them. In particular, it was necessary to extend the Hill approximation to treat simultaneous competition for cooperative as well as independent binding sites, while earlier attempts are shown to be inadequate. Criteria are developed for a qualitative analysis of complex binding patterns using the Scatchard-plot of the experimental data in order to obtain a model of the binding structure and an adequate set of input parameters for the numerical analysis. Numerical examples refer to the binding of calcium and magnesium to the sarcoplasmic reticulum as studied by competitive replacement of manganese ions [3].

Binding Sites↗