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H R Kalbitzer

Publications and source records attributed to H R Kalbitzer.

At least 73 records · Page 4Linked to original sources
PubMed1993

Oligomerization of the Nef protein from human immunodeficiency virus (HIV) type 1.

The nef genes, derived from two different human immunodeficiency-virus-type-1 (HIV-1) strains, were expressed in procaryotic cells (Escherichia coli) and in eucaryotic cells (insect cells infected with nef-containing baculovirus). The oligomerization of recombinant Nef protein was studied by NMR spectroscopy and immunoblotting under various experimental conditions. 1H-NMR spectroscopy shows that native folded protein has the tendency to polymerize under low-salt conditions. These oligomers become covalently linked by disulfide bonds after decreasing the reduction potential, a process which is fully reversible. Cross-linking studies with bis(sulfo-succinimidyl)suberate and alkylation with iodoacetic acid under non-reducing and reducing conditions document for the first time that Nef can also form homomeric structures including monomers, dimers, trimers and tetramers in cell lysates and intact cells. We found disulfide-linked as well as non-covalently associated oligomers. Since the Nef molecules are not exclusively found in the cytoplasm of HIV infected cells and since the reduced glutathione concentration in lymphocytes of virus infected persons is known to be unusually low, it might be possible that these Nef oligomers have a biological function in vivo as well.

Alkylation↗
PubMed1993

Involvement of various amino- and carboxyl-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus: site-directed mutagenesis with the ptsH gene, biochemical characterization and NMR studies of the mutant proteins.

The phosphocarrier HPr (heat stable protein) of Staphylococcus carnosus was modified by site-directed mutagenesis of the corresponding ptsH gene in order to analyse the importance of amino acids which were supposed to be part of the active centre of the protein. Three residues which are conserved in all HPrs, Arg17, Pro18 and Glu84, were mutated: Arg17 was changed to His (17RH) and Pro18 and Glu84 were changed into Ala (18PA and 84EA). In addition, Leu86 was changed into Ala (86LA) and one mutant protein was missing the last six residues of the HPr (delta 83). The wild type gene and all mutant genes were overexpressed and the gene products purified to homogeneity. Three-dimensional structures of wild type and mutant proteins were monitored by NMR spectroscopy. All five mutant HPrs had native conformations. The ATP-dependent HPr kinase can phosphorylate all HPr derivatives at Ser46. The PTS activity of the amino-terminal HPr mutant proteins 17RH and 18PA was different compared to wild type HPr. In contrast, the carboxy-terminal mutant HPrs possessed a similar enzyme activity to the wild type HPr. The 17RH and 18PA HPrs with substitution near the active centre His15 showed a very slow phosphorylation by enzyme I but the further transfer of the phosphoryl group to enzyme III was also strongly inhibited. The enzyme activity of the HPr 17RH was significantly improved at low pH. NMR pH-titration experiments showed that Arg17 is not responsible for the low pKa of the active centre His15 but this positively charged residue is essential in this position for the HPr activity.

Amino Acid Sequence↗
PubMed1993

Structure and function of proteins of the phosphotransferase system and of 6-phospho-beta-glycosidases in gram-positive bacteria.

New information about the proteins of the phosphotransferase system (PTS) and of phosphoglycosidases of homofermentative lactic acid bacteria and related species is presented. Tertiary structures were elucidated from soluble PTS components. They help to understand regulatory processes and PTS function in lactic acid bacteria. A tertiary structure of a membrane-bound enzyme II is still not available, but expression of Gram-positive genes encoding enzymes II can be achieved in Escherichia coli and enables the development of effective isolation procedures which are necessary for crystallization experiments. Considerable progress was made in analysing the functions of structural genes which are in close vicinity of the genes encoding the sugar-specific PTS components, such as the genes encoding the tagatose-6-P pathway and the 6-phospho-beta-glycosidases. These phosphoglycosidases belong to a subfamily of the beta-glycosidase family I among about 300 different glycosidases. The active site nucleophile was recently identified to be Glu 358 in Agrobacterium beta-glucosidase. This corresponds to Glu 375 in staphylococcal and lactococcal 6-phospho-beta-galactosidase. This enzyme is inactivated by mutating Glu 375 to Gln. Diffracting crystals of the lactococcal 6-P-beta-galactosidase allow the elucidation of its tertiary structure which helps to derive the structures for the entire glycosidase family 1. In addition, a fusion protein with 6-phospho-beta-galactosidase and staphylococcal protein A was constructed.

Amino Acid Sequence↗
PubMed1992

Expression, purification and biochemical characterisation of the human immunodeficiency virus 1 nef gene product.

The human immunodeficiency virus 1 (HIV-1) nef gene encoded by the HIV-1 isolate lymphadenopathy-associated virus type 1 was expressed in Escherichia coli under the control of the tac promoter. The protein is found mainly in the soluble part of the bacterial lysate; a simple two-column purification scheme has been developed allowing isolation of the recombinant protein without using denaturing agents. Analysis of the circular dichroism spectra reveals that the purified protein is folded and has a helix content of 16% and a beta-pleated sheet content of 31%. GTPase activity and binding of guanine nucleotides were measured for Nef and compared with the results obtained under identical experimental conditions for p21rasC, which represents a typical, well-characterized guanine-nucleotide-binding (GNB) protein. Within the limits of error, native Nef does not show GTPase activity and does not bind guanine nucleotides strongly (association constant, Kass less than 5 x 10(3) M-1). An upper limit for the association constant of Nef for ATP was determined by equilibrium dialysis as 5 x 10(3) M-1. Nef can be autophosphorylated by ATP; under the experimental conditions used, 1-2% of the protein become phosphorylated. Correspondingly, our Nef preparation shows a low, but significant, ATPase activity. In conclusion, Nef is not a member of the GNB protein family, but a possible role as a protein kinase cannot be excluded.

Adenine Nucleotides↗
PubMed1992

High protein mobility in skinned rabbit muscle fibres observed by 1H NMR spectroscopy.

1H NMR spectra of skinned rabbit muscle fibers show a group of relatively sharp resonance lines which presumably originate from highly mobile protein domains. Comparison with the spectrum of myosin subfragment 1 suggests that these signals may come at least partly from mobile regions of the myosin head. NMR could possibly be used to characterize the movements of crossbridges in force generation.

Animals↗
PubMed1992

Biochemical analysis of cervical mucus by nuclear magnetic resonance spectroscopy.

Biochemical evaluation of cervical mucus is difficult due to the characteristic rheological properties of this hydrogel. The application of high resolution nuclear magnetic resonance spectroscopy proved to be a valuable new method for differentiated biochemical analyses of human cervical mucus. A particular advantage is that it is non-destructive, that it can be applied to specimens of small volume and that no sample preparation, such as solubilization, is necessary.

Cervix Mucus↗
PubMed1991

Two-dimensional 1H NMR studies on HPr protein from Staphylococcus aureus: complete sequential assignments and secondary structure.

Complete sequence-specific assignments of the 1H NMR spectrum of HPr protein from Staphylococcus aureus were obtained by two-dimensional NMR methods. Important secondary structure elements that can be derived from the observed nuclear Overhauser effects are a large antiparallel beta-pleated sheet consisting of four strands, A, B, C, D, a segment SAB consisting of an extended region around the active-center histidine (His-15) and an alpha-helix, a half-turn between strands B and C, a segment SCD which shows no typical secondary structure, and the alpha-helical, C-terminal segment S(term). These general structural features are similar to those found earlier in HPr proteins from different microorganisms such as Escherichia coli, Bacillus subtilis, and Streptococcus faecalis.

Amino Acid Sequence↗
PubMed1991

C-terminal structure and mobility of rabbit skeletal muscle light meromyosin as studied by one- and two-dimensional 1H NMR spectroscopy and X-ray small-angle scattering.

Intact rabbit myosin and two different C-terminal fragments of rabbit muscle light meromyosin (LMM) expressed in Escherichia coli, LMM-30, and LMM-30C', were studied by 1H NMR spectroscopy. X-ray small-angle scattering shows that at high ionic strength two polypeptide chains of LMM-30 (which consists of the C-terminal 262 amino acids of myosin heavy chain) or LMM-30C' (which corresponds to LMM-30 but lacks the last 17 residues) assemble to form an alpha-helical coiled-coil as it is found also in myosin. The last 12 C-terminal residues of one polypeptide chain of LMM-30 and the last 9 C-terminal residues of the other chain are very mobile. The last 8 residues of the two strands are equivalent from the NMR point of view and unfolded; the valine residues in position 255 in the two strands are not equivalent, suggesting an interaction between the two strands, Ser-252, Arg-253, and Asp-254 are completely immobilized in one of the polypeptide strands and partly mobile in the other. Essentially the same pattern is observed in intact myosin. In spite of the large molecular weights of LMM-30 and LMM-30C', it is possible to resolve almost all aromatic residues and to determine the pK values of all the 4 tyrosine and of 9 (out of 10) histidine residues. The tyrosine residues in the two strands are equivalent in the two polypeptide chains and both have a pK of 10.5. The pK values of the histidine residues vary between 5.7 and 7.0.

Amino Acid Sequence↗
PubMed1991

The effect of kirromycin on the metal ion coordination in complexes of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction.

The binding of the antibiotic kirromycin to elongation factor Tu (EF-Tu) leads to a severe line broadening of the Q-band manganese EPR lines of EF-Tu.Mn, EF-Tu.Mn.GDP, as well as EF-Tu.Mn.GDP.Pi indicating that the coordination sphere of the metal ion is changed by this interaction. The number of coordinated water molecules was determined from the 17O-55Mn superhyperfine coupling observable in H2(17)O enriched water; it is pH-dependent in the EF-Tu.Mn.GDP complex, at pH 6.8 probably four water molecules are coordinated with the protein bound manganese, at pH 8.4 one of the water molecules is replaced by a functional group from the protein. Independent of the pH, probably four and five water molecules are bound to the metal ion in the EF-Tu.Mn and in the EF-Tu.Mn.GDP.Pi complex, respectively. Kirromycin does not influence the number of water molecules bound to EF-Tu.Mn.GDP and EF-Tu.Mn.GDP.Pi, but leads to an increase of the number of water molecules coordinated to the metal ion in EF-Tu.Mn. The 17O-55Mn superhyperfine coupling constant in Mn(H2O)6 was determined from the EPR-spectra as 0.24 mT.

Anti-Bacterial Agents↗
PubMed1991

Rabbit skeletal muscle myosin. Unfolded carboxyl-terminus and its role in molecular assembly.

We have expressed in E. coli segments of the rod portion of rabbit skeletal fast muscle myosin and compared physical properties of two different species, LMM-30 and LMM-30C'. LMM-30 consists of 263 amino acids including the original C-terminus of myosin heavy chain. LMM-30C' is colinear with LMM-30, but is devoid of 17 residues at the C-terminus. 1H NMR spectroscopy indicates that the C-terminus of LMM-30, but not of LMM-30C' is unfolded and freely mobile. Furthermore, the present results show that the unfolded C-terminus is essential for molecular assembly of LMM-30; at pH 8.0 LMM-30, but not LMM-30C', formed aggregates upon decreasing the ionic strength.

Amino Acid Sequence↗
PubMed1991

Enzyme IIIlac of the staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis of histidine residues, biochemical characterization and 1H-NMR studies.

The lactose-specific phosphocarrier protein enzyme III of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacF gene in order to replace the histidine residues 78 and 82 of the amino acid sequence with a serine residue. Wild-type and both mutant genes were overexpressed in Escherichia coli and the gene products were purified to homogeneity. The conformation of wild-type and mutant proteins were monitored by 1H-NMR spectroscopy. In vitro phosphorylation studies on mutant lactose-specific enzyme III, as well as evidence from NMR spectroscopy, lead to the conclusion that His78 is the active-site for phosphorylation of lactose-specific enzyme III by phospho-HPr (histidine-containing protein). The role of His82 probably is the enhancement of velocity and efficiency of the phosphotransfer from lactose-specific enzyme III to lactose-specific enzyme II. This result refutes the conclusion of former work based on data by protelytic cleavage and sequencing of the 32P-labeled peptide of lactose-specific enzyme III that His82 is the active-site for phosphorylation.

Bacterial Proteins↗
PubMed1990

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction.

Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF-Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)-(Rp)-[beta-17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line-broadening of the EPR spectrum of Mn2+ at the active site of EF-Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF-Tu.GDP.Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2'(3')-O-(N-methylanthraniloyl)-GDP is 6.2 x 10(-3) s-1 at 25 degrees C and pH 6.8], we conclude that the broadened signal arises from the EF-Tu.Mn.GDP.Pi complex, the predominant steady-state species. During the hydrolysis of GTP the Mn2+ remains bound to the beta-phosphate oxygen of GDP which arises from the beta pro-S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF-Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)-[beta-17O]GTP in the corresponding complex in the presence of EF-Ts.

Binding Sites↗
PubMed1989

1H-nuclear magnetic resonance studies of the neuropeptide head activator.

The 1H-NMR spectrum of the neuropeptide head activator in aqueous solution has been completely assigned by two-dimensional NMR spectroscopy and selective deuteration. The apparent pseudo-first-order exchange rate, kex, of the backbone amide protons and the correspondent activation enthalpies, delta H not equal to, were determined. The exchange rates decrease and the activation enthalpies increase from the N-terminal to the C-terminal part of the peptide. The exchange rates vary from 21 to 0.3 s-1 at 274 K, the activation enthalpies from 60 to 75 kJ.mol-1. The pK values of the terminal carboxyl group and of the lysine amino group have been estimated as 3.3 and 10.3, respectively. The NMR results are in line with a dimeric structure in an antisymmetric arrangement of the subunits, forming an antiparallel beta-pleated sheet between C-terminal segments. The peptide bonds between pGlu-1, Pro-2 and Pro-3 are predominantly in trans-configuration, in fact no cis-isomers can be observed spectroscopically. The structure appears to be very stable; in the temperature and pH range studied, i.e., from 274 to 338 K and from pH 0.8 to pH 11.6, there are no spectroscopic indications for a global structural change.

Hot Temperature↗
PubMed1987

Phosphorylation of the sarcoplasmic calcium-activated adenosinetriphosphatase as studied by 31P nuclear magnetic resonance.

A reinvestigation of a study of Fossel et al. [Fossel, E. T., Post, R. L., O'Hara, D.S., & Smith, T. W. (1981) Biochemistry 20, 7215-7219] in which the 31P nuclear magnetic resonance (NMR) signal of the phosphointermediate of the sarcoplasmic (Ca2+, Mg2+)-ATPase has been identified shows that the signal they describe most probably originates from free Mg . ATP but not from the phosphoenzyme itself. It was possible to detect the 31P NMR signal of the phosphoenzyme in peptic fragments of sarcoplasmic ATPase phosphorylated either by ATP or by inorganic phosphate. The two products exhibit the same spectral characteristics in 31P NMR, implying that most probably both reaction pathways yield the same chemical product. Chemical shifts at low pH (-6.5 ppm) and high pH (-1.4 ppm) of the phosphoryl group are indicative of a beta-phosphoaspartyl moiety, thus confirming independently the results from chemical analysis. The relatively low pK value of 4.3 of the phosphoryl group suggests an interaction with a positively charged group of the enzyme.

Animals↗
PubMed1987

Characterisation of the metal-ion-GDP complex at the active sites of transforming and nontransforming p21 proteins by observation of the 17O-Mn superhyperfine coupling and by kinetic methods.

Kinetic studies on the interaction of three Ha-ras-encoded p21 proteins with GDP and MgGDP have yielded values for the association (10(6)-10(7) M-1 s-1) and dissociation (10(-3)-10(-5) s-1) rate constants at 0 degrees C. Dramatic differences in the rate constants were not observed for the three proteins. Under non-physiological conditions (absence of Mg2+), the rate constant for GDP release was an order of magnitude faster for the viral protein p21v than for the cellular form p21c or the T24 mutant p21t, but this was reduced to a factor of about 3 in the presence of Mg2+. In all cases, there was an increase of about one order of magnitude in the rate of GDP release on removing magnesium. The binding affinities ranged from 5.7 X 10(10) M-1 for p21c to 1.3 X 10(11) M-1 for p21v. Electron paramagnetic resonance (EPR) measurements on Mn2+ bound together with stereospecifically 17O-labelled GDP showed direct coordination of a beta-phosphate oxygen to the metal ion with a superhyperfine coupling constant of 0.16-0.22 mT, but no interaction with the alpha-phosphate oxygens at the active site of all three proteins. The association constant of Mn(II) to p21 proteins in the absence of nucleotides was estimated to be greater than 10(5) M-1. In agreement with the EPR results, experiments on the metal ion dependence of the binding of thiophosphate analogs of GDP provided further evidence for the absence of direct coordination of the metal ion to the alpha-phosphate group. These results have been used to construct a model for the interactions of Mg X GDP with the active site of p21 proteins.

Binding Sites↗
PubMed1986

Observation of Mg2+.ATP and uncomplexed ATP in slow exchange by 31P-NMR at high magnetic fields.

The 31P-NMR lines of the beta-phosphate groups in Mg2+.ATP and in metalfree ATP can be observed separately up to 280 K at 8.5 T and up to 285 K at 11.7 T. At 274 K and 8.5 T the beta-phosphorous resonances are in slow exchange at pH values above pH 5, the gamma-phosphorous resonances are in slow exchange only near pH 6, but in fast exchange at low and high pH-values. The fast exchange condition holds for the alpha-phosphorous resonances over the entire pH-range. For Ca2+.ATP and metalfree ATP always fast exchange prevails down to the freezing point of water even at 11.7 T. Based on the separate observation of the 31P-NMR signals of Mg2+.ATP and uncomplexed ATP new experiments are proposed and possible sources of error in 'in vivo' NMR studies are discussed.

Adenosine Triphosphate↗
PubMed1985

Phosphoenolpyruvate-dependent phosphotransferase system. 1H NMR studies on chemically modified HPr proteins.

The low-pK tyrosyl residue present in the heat-stable proteins (HPr) of all Gram-positive bacteria studied until now has been labeled by tetranitromethane in the HPr of Bacillus subtilis and Streptococcus faecalis. The nitrotyrosyl derivatives obtained are fully active in the complementation assay. The labeled tyrosyl residues could be identified as Tyr-37 in both proteins. Reinvestigation of the low-pK tyrosyl residue in HPr of Staphylococcus aureus resulted in the same assignment. In all three proteins an interaction between nitrotyrosine-37 and the active center His-15 could be observed, leading to an increase in the pK of His-15 and a change of its chemical shift parameters. The 1H NMR lines of the complete aromatic spin system of HPr of B. subtilis could be assigned by the nitration studies. Labeling of Arg-17 in HPr of S. aureus and S. faecalis by 1,2-cyclohexanedione in the presence of borate ions causes an almost complete inhibition of its enzymatic activity. In the NMR spectrum the labeling of the arginyl residue influences the resonance lines of His-15: two new resonance lines for the C-2 protons of equal intensity are observed, a fact that could be explained by two different conformations in slow exchange. The pK value of His-15 was not changed by the labeling, excluding Arg-17 as responsible for the low pK of His-15.

Amino Acid Sequence↗