Biomedical subjects

G Otting

Publications and source records attributed to G Otting.

81 records · Page 5Linked to original sources

PubMed1989

The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors.

The structure of the Antennapedia homeodomain from Drosophila melanogaster was determined by nuclear magnetic resonance spectroscopy in solution. It includes three well-defined helices (residues 10-21, 28-38, and 42-52) and a more flexible fourth helix (residues 53-59). Residues 30-50 form a helix-turn-helix motif virtually identical to those observed in various prokaryotic repressors. Further comparisons of the homeodomain with prokaryotic repressors showed that there are also significant differences in the molecular architectures. Overall, these studies support the view that the third helix of the homeodomain may function as the DNA recognition site. The elongation of the third helix by the fourth helix is a structured element that so far appears to be unique to the Antennapedia homeodomain.

Animals↗

PubMed1989

Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling.

Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.

Carbon Isotopes↗

PubMed1988

Isolation and sequence-specific DNA binding of the Antennapedia homeodomain.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was overproduced in a T7 expression vector in Escherichia coli. The corresponding polypeptide of 68 amino acids was purified to homogeneity. The homeodomain was analysed by ultracentrifugation and assayed for DNA binding. The secondary structure of the isolated homeodomain was determined by nuclear magnetic resonance spectroscopy. DNA-binding studies indicate that the isolated homeodomain binds to DNA in vitro. It selectively binds to the same sites as a longer Antp polypeptide and a full-length fushi tarazu (ftz) protein. Therefore, the homeodomain represents the DNA-binding domain of the homeotic proteins.

Animals↗

PubMed1988

Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was studied in aqueous solution by nuclear magnetic resonance (NMR). Sequence-specific resonance assignments have been obtained for the complete polypeptide chain of 68 amino acid residues. The secondary structure determined from nuclear Overhauser effects (NOE) and information about slowly exchanging amide protons includes three helical segments consisting of the residues 10-21, 28-38 and 42-52, respectively. Combination of the presently available NMR data with computer modeling provided preliminary evidence for the presence of a helix-turn-helix motif in the homeodomain. Near the turn, this supersecondary structure appears to be very similar to the DNA binding site in the 434 and P22 c2 repressors, but both helices in the homeodomain include 2-3 additional residues when compared with these prokaryotic DNA-binding proteins.

Amino Acid Sequence↗

PubMed1988

OR3 operator of bacteriophage lambda in a 23 base-pair DNA fragment: sequence-specific 1H NMR assignments for the non-labile protons and comparison with the isolated 17 base-pair operator.

Sequence-specific 1H NMR assignments are presented for a non-selfcomplementary 23-base-pair DNA duplex of molecular weight 15,000 daltons, containing the OR3 repressor binding site of bacteriophage lambda as the central core. The NMR techniques used were mainly phase-sensitive two-dimensional NOE and 2Q spectroscopy, the latter to overcome overlap problems within the spectral region of the deoxyribose spin-systems. Direct sequential NOE connectivities are observed between adenine 2 H and deoxyribose 1' protons. We propose the use of these connectivities as a check of the assignments of C1' and A2 protons, which have independently been derived via other assignment pathways.

Bacteriophage lambda↗

PubMed1987

Monitoring the purification by high-performance liquid chromatography of cardiotoxins from Naja mossambica mossambica using phase-sensitive two-dimensional nuclear magnetic resonance.

High-resolution phase-sensitive two-dimensional proton nuclear magnetic resonance was used to monitor the preparation by high-performance liquid chromatography of homogeneous proteins from the venom of Naja mossambica mossambica. This resulted in the characterization of a heterogeneous protein preparation VII2, which had been used in earlier structural studies by NMR, as well as a homogeneous protein CTXIIb and a nearly homogeneous protein fraction CTXIIa, which are now both subject to further investigations of their solution conformations.

Amino Acids↗

PubMed1987

Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica.

Sequence-specific assignments of the 1H-nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two-dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25 degrees C and 45 degrees C for all but one backbone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side-chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497-508]. The 1H/2H-exchange kinetics were measured in 2H2O at 20 degrees C for the amide protons and the N-terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double-stranded antiparallel beta-sheet comprising the residues 2-4 and 11-13, and a triple-stranded antiparallel beta-sheet consisting of the residues 20-26, 35-39, and 49-55. The two peripheral strands of the triple-stranded beta-structure were found to be connected by a right-handed cross-over, and the locations of several tight turns were also identified.

Amino Acid Sequence↗

PubMed1987

Sequential NMR assignments of labile protons in DNA using two-dimensional nuclear-Overhauser-enhancement spectroscopy with three jump-and-return pulse sequences.

Two-dimensional nuclear Overhauser enhancement (NOESY) spectra of labile protons were recorded in H2O solutions of a protein and of a DNA duplex, using a modification of the standard NOESY experiment with all three 90 degree pulses replaced by jump-and-return sequences. For the protein as well as the DNA fragment the strategically important spectral regions could be recorded with good sensitivity and free of artifacts. Using this procedure, sequence-specific assignments were obtained for the imino protons, C2H of adenine, and C4NH2 of cytosine in a 23-base-pair DNA duplex which includes the 17-base-pair OR3 repressor binding site of bacteriophage lambda. Based on comparison with previously published results on the isolated OR3 binding site, these data were used for a study of chain termination effects on the chemical shifts of imino proton resonances of DNA duplexes.

Base Composition↗

PubMed1987

15N-labeled P22 c2 repressor for nuclear magnetic resonance studies of protein-DNA interactions.

The salmonella phage P22 c2 repressor was produced with 90% 15N isotope labeling of all leucines, using the expression system E. coli W3110 lac IQ¿P 125. The N-terminal DNA-binding domain 1-76 was obtained by chymotrypsin cleavage. Its characterization by biochemical techniques, mass spectrometry, and one- and two-dimensional nuclear magnetic resonance (NMR) showed that highly residue-selective isotope labeling was achieved with the minimal growth medium used. The ability to obtain such isotope labeling opens new avenues for NMR studies of protein-DNA interactions in the P22 operator system.

Escherichia coli↗