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Biomedical subjects

A Wittinghofer

Publications and source records attributed to A Wittinghofer.

At least 181 records · Page 10Linked to original sources
PubMed1986

Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site.

v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.

Cell Line↗
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↗
PubMed1983

Stereochemistry of the elongation factor Tu X GTP complex.

The geometry of the Me2+. GTP complex at the active site of EF-Tu from Bacillus stearothermophilus has been investigated using thiophosphate analogs of GTP to inhibit the kirromycin-induced GTPase reaction at 60 mM NH4Cl. There is no reversed selectivity for the diastereomers (Rp and Sp) of guanosine 5'-O-(1-thiotriphosphate) (GTP[alpha S]) on replacing Mg2+ by Cd2+, so that the observed specifity for the Sp isomer must be due to an interaction of the pro-R oxygen of the alpha-phosphate group with the protein. With the diastereomers of GTP[beta S] low specifity for the Rp isomers is seen in the presence of Mg2+. Moreover, both isomers are very weakly bound. In contrast, substitution of Mg2+ by Cd2+ results in a high specifity for the Sp isomer, and this is then recognized as well as Cd X GTP. These results indicate that in the EF-Tu X Me2+ X GTP complex, the pro-S oxygen of the beta-phosphate group is bound to the metal ion and the pro-R oxygen to the protein. GTP[gamma S] is a good analog of GTP regardless of the nature of the metal ion, suggesting that not all of the oxygens of the gamma-phosphate are involved in interactions to metal ion and protein. The thiophosphate analogs of GTP were also tested for their efficiency in ternary complex formation with EF-Tu and aminoacyl-tRNA and in the physiological GTPase of EF-Tu. The stereochemistry of the GTP binding site on EF-Tu in all three systems is found to be very similar.

Binding Sites↗
PubMed1983

Bacterial elongation factor Ts: isolation and reactivity with elongation factor Tu.

An improved method for the purification of bacterial polypeptide elongation factor Ts (EF-Ts) from one mesophile (Escherichia coli) and two thermophiles (Bacillus stearothermophilus and PS3) is described. The improvements are both in the facility of isolation and in increased yields. The purified factors were used for cross-reactivity studies with elongation factor Tu (EF-Tu) obtained from the same bacterial strains. In all combinations studied, the efficiency of EF-Ts in catalyzing the exchange of EF-Tu-bound GDP was proportional to the strength of the protein-protein complex. Whereas the factors from the two thermophiles were interchangeable, the mesophilic EF-Ts formed a very weak complex with thermophilic EF-Tu; however, thermophilic EF-Ts formed very strong complexes with mesophilic EF-Tu. Thus, e.g., EF-Tu from E. coli formed a complex with EF-Ts from B. stearothermophilus which was 10 times more stable than the corresponding homologous complex.

Guanosine Diphosphate↗
PubMed1982

The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP.

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.

Escherichia coli↗
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

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↗
PubMed1980

Composition and properties of trypsin-cleaved elongation factor Tu.

Native elongation factor Tu from Escherichia coli, EF-Tu, is initially attacked by trypsin at three adjacent sites in the primary structure. These are arginine-44, arginine-58, and lysine-56. The rates of hydrolysis at the two arginine residues are about the same but that at the lysine residue is much slower. The products of the tryptic digestion have been analysed by Edman degradation and polyacrylamide gel electrophoresis. The peptide from alanine-45 to arginine-58 is eventually excised and does not complex with the remaining polypeptides (fragments A and D). The loss of this peptide does not lead to a concomitant loss of activity in stimulating polyphenylalanine synthesis. The latter is closely correlated with the further hydrolysis of the remaining fragment (A + D) complex. This complex resembles native EF-Tu in its ability to stimulate both polyphenylalanine synthesis and the binding of aminoacyl-tRNA to 70-S ribosomes, but does not form so stable a ternary complex with aminoacyl-tRNA and GTP as the native protein.

Amino Acid Sequence↗
PubMed1979

The effect of Mg2+ on some properties of nucleotide-free elongation factor Tu from Bacillus stearothermophilus.

The nucleotide-free elongation factor from Bacillus stearothermophilus provides a means to study the effect of Mg2+ ions on various reactions of the protein. The binding of GDP to the protein is stimulated by Mg2+. From comparative studies with other metal ions, particularly Mn2+, it appears that this stimulation is due to the formation of a metal - GDP complex which is bound to the protein. Protection against proteolysis by trypsin is afforded by both Mg2+ and Mg - GDP, but not by GDP alone. The rate of substitution of the sulphydryl group associated with aminoacyl-tRNA binding, either 5,5'-dithio-bis(2-nitrobenzoic acid) or N-ethylmaleimide is reduced in the presence of Mg2+ - All these observations show that Mg2+ not only is involved in GDP binding but also has a direct effect on the tertiary structure of the protein.

Calcium↗
PubMed1976

Elongation factor T from Bacillus stearothermophilus and Escherichia coli. Purification and some properties of EF-Tu and EF-Ts from Bacillus stearothermophilus.

Homogeneous preparations of elongation factors EF-Tu and EF-Ts from Bacillus stearothermophilus have been obtained with specific activities of 20000 +/- 2000 and 500000 +/- 50000 units/mg, respectively. By dodecylsulphate-polyacrylamide gel electrophoresis the molecular weight of EF-Tu was found to be 49000 +/- 2000 and of EF-Ts 35500 +/- 1000. Nucleotide-free EF-Tu was prepared by using ITP as a GDP-binding-site-directed analogue. EF-Tu was shown to contain two sulphydryl groups, one reacting fast and one slowly with N-ethylmaleimide and 5,5'-dithio-bis(2-nitrobenzoic acid) under non-denaturing conditions. The same reagents were shown to react with the three sulphydryl groups of EF-Ts in the native state. The heat stabilities of EF-Tu and EF-Ts are reversed with respect to the Escherichia coli factors, EF-Tu being the more stable protein; even nucleotide-free EF-Tu is relatively stable with a half-life at room temperature of about 35 h.

Amino Acids↗