Biomedical subjects

H Schwalbe

Publications and source records attributed to H Schwalbe.

25 records · Page 2Linked to original sources

PubMed1996

Directed TOCSY, a method for selection of directed correlations by optimal combinations of isotropic and longitudinal mixing.

The directed TOCSY pulse sequence element transfers coherence predominantly into "forward-directed" antiphase coherences while simultaneously suppressing in-phase and "backward-directed" antiphase coherences. This novel selection principle, based on the "direction" of the target coherences, provides a new approach for the simplification of crowded spectra. In this article, the theory of directed TOCSY is presented for linear spin systems that are frequently found in carbon-labeled biomolecules.

Magnetic Resonance Imaging↗

PubMed1996

The concept of a random coil. Residual structure in peptides and denatured proteins.

Non-native states of proteins are of increasing interest because of their relevance to issues such as protein folding, translocation and stability. A framework for interpreting the wealth of experimental data for non-native states emerging from rapid advances in experimental techniques involves comparison with a "random coll' state, which possesses no structure except that inherent in the local interactions. We review here the concept of a random coil, from its global to its local properties. In particular, we focus on the description of a random coil in terms of statistical distributions in psi, phi space. We show that such a model, in combination with experimental data, provides insight into the structural properties of polypeptide chains and has significance for understanding protein folding and for molecular design.

Animals↗

PubMed1995

Characterization of conformational preferences in a partly folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg-white lysozyme in trifluoroethanol.

2D and 3D heteronuclear NMR methods have been used to characterize the structure of hen egg-white lysozyme in a partially folded state using uniformly 15N-labeled protein. This state is formed by the denaturation of the protein in 70% trifluoroethanol (TFE)/30% water (v/v) and is characterized by substantial helical secondary structure in the absence of extensive tertiary interactions. 15N-filtered 3D NOESY and TOCSY experiments have allowed the sequential assignment of resonances for all but 2 of the 126 main chain amide nitrogen atoms and of the majority of main and side chain proton resonances. The conformation of the polypeptide chain was characterized by analysis of the pattern of NOEs, H alpha chemical shift perturbations, 3J(HN, H alpha)-coupling constants, and hydrogen exchange protection. These NMR parameters are highly complementary and are consistent with a model for the TFE state in which six regions of the polypeptide chain are substantially ordered in helical conformations. The structure in different regions however, shows different levels of persistency. Five of the helices exhibit significant protection of amide hydrogens against exchange with solvent and are located in regions of the polypeptide which are helical in the native state. By contrast, helical structures of greater flexibility are observed both as extensions to the native-like helices and as a nonnative structure in the region of the molecule which forms the C-terminal part of the beta-sheet in the native state. No specific structural preferences are detected in regions corresponding to the long loop and to the N-terminal part of the beta-sheet of native lysozyme. A combination of local features of the polypeptide chain, including the predicted propensities of residues for helix formation and for their participation in N- and C-terminal helix capping interactions, allows the conformational behavior of the polypeptide chain of hen lysozyme to be rationalized for this partially folded state. The analysis implies that the nonnative structures are a result of interactions which are local to the polypeptide chain. These, and the highly persistent native-like structures, give insight into species which form early during folding.

Amides↗

PubMed1995

Sequential correlation of anomeric ribose protons and intervening phosphorus in RNA oligonucleotides by a 1H, 13C, 31P triple resonance experiment: HCP-CCH-TOCSY.

A three-dimensional 1H, 13C, 31P triple resonance experiment, HCP-CCH-TOCSY, is presented which provides unambiguous through-bond correlation of all 1H ribose protons on the 5' and 3' sides of the intervening phosphorus along the backbone bonding network in 13C-labeled RNA oligonucleotides. The correlation of the complete ribose spin system to the intervening phosphorus is obtained by adding a C,C-TOCSY coherence transfer step to the triple resonance HCP experiment. The C,C-TOCSY transfer step, which utilizes the large and relatively uniform 1J(C,C) coupling constant (approximately 40 Hz for ribose carbons), efficiently correlates the phosphorus-coupled carbons observed in the HCP correlation experiment (i.e., C4' and C5' in the 5' direction and C4' and C3' in the 3' direction) to all other carbons in the ribose spin system. Of the additional correlations observed in the HCP-CCH-TOCSY, that to the relatively well-resolved anomeric H1',C1' resonance pairs provides the greatest gain in terms of facilitating assignment. The gain in spectral resolution afforded by chemical shift labeling with the anomeric resonances should provide a more robust pathway for sequential assignment over the intervening phosphorus in larger RNA oligonucleotides. The HCP-CCH-TOCSY experiment is demonstrated on a uniformly 13C, 15N-labeled 19-nucleotide RNA stem-loop, derived from the antisense RNA I molecule found in the ColE1 plasmid replication control system.

Base Sequence↗

PubMed1994

Conformation of valine side chains in ribonuclease T1 determined by NMR studies of homonuclear and heteronuclear 3J coupling constants.

A conformational analysis of the valine side chains of ribonuclease T1 (RNase T1) was performed using NMR spectroscopy, in particular homonuclear (1H, 1H and 13C, 13C) and heteronuclear (1H, 15N and 1H, 13C) vicinal spin-spin coupling constants as obtained from E.COSY-type NMR experiments. The coupling constants related to the chi 1 dihedral angle in valine, 3JH alpha H beta, 3JNH beta, 3JC'H beta, 3JH alpha C gamma 1, 3JH alpha C gamma 2, 3JC'C gamma 1, and 3JC'C gamma 2, were evaluated in a quantitative manner. The analysis of 3J data allowed for the stereospecific assignment of the valine methyl resonances. On the basis of various models for motional averaging of coupling constants, a fit of the torsion angles chi 1 to a set of the experimental 3J coupling constants (3JH alpha H beta, 3JNH beta, 3JC'H beta) was carried out. The resulting side-chain conformations were examined with respect to NOE distance informations. Single rotameric states emerged for Val16, Val67, Val79, and Val101, while conformational equilibria between staggered rotamers were found for Val33 and Val78. Using a different model approach, Val52 and Val89 are also likely to exhibit unimodal chi 1 angle distributions. The analysis was found to depend critically on the set of Karplus parameters used. Except for Val52 and Val78, the predominant rotamers obtained from 3J coupling informations agree with the conformation in the crystal structure of ribonuclease T1 (Martinez-Oyanedel et al., 1991).

Magnetic Resonance Spectroscopy↗

PubMed1994

Determination of a complete set of coupling constants in 13C-labeled oligonucleotides.

Three experiments are introduced to determine a complete set of coupling constants in RNA oligomers. In the HCCH-E.COSY experiment, the vicinal proton-proton coupling constants can be measured with high accuracy. In the P-FIDS-CT-HSQC experiment, vicinal proton-phosphorus and carbon-phosphorus couplings are measured that depend on the phosphodiester backbone torsion angles beta and epsilon. In the refocussed HMBC experiment, vicinal carbon-proton couplings are measured that depend on the glycosidic torsion angle chi.

Base Sequence↗

PubMed1992

Secretion of mammalian ribonucleases from Escherichia coli using the signal sequence of murine spleen ribonuclease.

A nucleotide sequence identical with that of the recently identified murine pancreatic ribonuclease (RNAase) was isolated from a murine spleen cDNA library. Active RNAase was expressed and secreted from Escherichia coli lon-htpr- transformed with a plasmid containing the E. coli trp promoter followed by the murine RNAase gene sequence, including the original eukaryotic 26-amino-acid signal sequence. Approx. 1 mg of properly matured RNAase protein/litre was secreted into the medium of a fermentor culture after the promotor was induced by tryptophan starvation. When the signal sequence was deleted from the plasmid, intracellular RNAase activity was very low and there was no significant supernatant RNAase activity. Even higher RNAase yields were obtained with a synthetic gene for bovine pancreatic ribonuclease cloned after the signal sequence of the murine gene. About 2 mg of correctly processed RNAase A/litre was isolated from the growth medium, and a further 8-10 mg of correctly processed RNAase/litre could be isolated from the soluble fraction of the cells. Thus this eukaryotic signal sequence is both recognized by the E. coli transport and processing apparatus and gives efficient secretion, as well as export, of active, mature mammalian RNAases.

Amino Acid Sequence↗