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Christian Griesinger

Publications and source records attributed to Christian Griesinger.

49 records · Page 3Linked to original sources
PubMed2003

Dipolar couplings in multiple alignments suggest alpha helical motion in ubiquitin.

Recently, residual dipolar couplings (RDCs) of backbone N-HN vectors measured in 11 different alignment media were analyzed with respect to structure and dynamics in a model-free way in terms of generalized order parameters and motional anisotropies. The anisotropies in the central alpha-helix were found to be strikingly uniformly distributed. In this communication, these parameters are further interpreted in terms of physically feasible cooperative reorientational motion of the helix with respect to the core of the protein. The RDCs are compatible with a model in which all N-HN vectors of the alpha-helix of ubiquitin exhibit correlated anisotropic excursions with amplitudes of 21 degrees and 12 degrees along two orthogonal directions x' ' and y' ' of a coordinate system C' ', if z' ' represents the helix axis. Such motion contradicts neither NOE data nor molecular force-field calculations.

Models, Chemical↗
PubMed2003

Self-consistency analysis of dipolar couplings in multiple alignments of ubiquitin.

A self-consistency analysis of backbone N-H residual dipolar couplings of ubiquitin collected in 10 different media is described to assess the degree of structural and dynamic heterogeneous behavior across the media. The SECONDA method, which works with and without any structural or dynamic information about the molecular system, is based on a principal component analysis and is very sensitive to the presence of heterogeneities or experimental errors. It is found that the regular secondary structural elements behave highly homogeneously, while small heterogeneities are manifested in the loop region 51-63. Many residues that exhibit increased dynamics in NMR relaxation experiments are inert with respect to changes in the alignment.

Models, Molecular↗
PubMed2003

Measurement of long range H,C couplings in natural products in orienting media: a tool for structure elucidation of natural products.

In this paper we show that water insoluble compounds dissolved in poly-gamma-benzyl-glutamate are amenable to the measurement of a number of homo- and heteronuclear dipolar couplings. The sensitivity and experimental precision of dipolar couplings are sufficient to obtain a good match with the structure. In order to achieve the necessary precision for H,C dipolar couplings between protons and carbons that are not directly bound a new method for the measurement of heteronuclear long range couplings is introduced that allows a one-parameter fit to a HSQC-based experiment as reference experiment. The methodology is applied to menthol (1R, 3S, 4R).

Carbon↗
PubMed2003

Measuring the chi 1 torsion angle in protein by CH-CH cross-correlated relaxation: a new resolution-optimised experiment.

Here we introduce an experiment with high sensitivity and resolution for the measurement of CH-CH dipolar-dipolar cross-correlated relaxation rates (CCRR) in protein side-chains. The new methodology aims to the determination of structural and dynamical parameters around the torsion angle chi(1) by measuring C(alpha)H(alpha)-C(beta)H(beta) cross-correlated relaxation rates. The method is validated on the protein ubiquitin: the chi(1) angles determined from the CCRR data are compared with the chi(1) angles of a previously determined NMR structure. The agreement between the two data sets is excellent for most residues. The few discrepancies that were found between the CCR-derived chi(1) angles and the angles of the previously determined NMR structure could be explained by taking internal motion into account. The new methodology represents a very powerful tool to determine both structure and dynamics of protein side-chains in only one experiment.

Magnetic Resonance Spectroscopy↗
PubMed2002

A structural model of the complex formed by phospholamban and the calcium pump of sarcoplasmic reticulum obtained by molecular mechanics.

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids that modulates the activity of the reticular Ca(2+) ion pump. We recently solved the three-dimensional structure of chemically synthesized, unphosphorylated, monomeric PLN (C41F) by high-resolution nuclear magnetic resonance spectroscopy in chloroform/methanol. The structure is composed of two alpha-helical regions connected by a beta turn (Type III). We used this structure and the crystallographic structure of the sarcoplasmic reticulum calcium pump (SERCA) recently determined by Toyoshima and co-workers and modeled into its E(2) form by Stokes (1KJU) or by Toyoshima (1FQU). We applied restrained and unrestrained energy optimizations and used the AMBER molecular mechanics force field to model the complex formed between PLN and the pump. The results indicate that transmembrane helix 6 (M6) of the SERCA pump is energetically favored, with respect to the other transmembrane helices, as the PLN binding partner within the membrane and is the only one of these helices that also permits contact between the N-terminal residues of PLN and the critical cytosolic binding loop region of the pump. This result is in agreement with published biochemical data and with the predictions of previous mutagenesis work on the membrane sector of the pump. The model reveals that PLN does not span the entire width of the membrane, that is, its hydrophobic C-terminal end is located near the center of the transmembrane region of the SERCA pump. The model also shows that interaction with M6 is stabilized by additional contacts made by PLN to M4. The contact between the N-terminal portion of PLN and the pump is stabilized by a number of salt and hydrogen-bond bridges, which may be abolished by phosphorylation of PLN. The contacts between the cytosolic portions of PLN and the pump are only observed in the E(2) conformation of the pump. Our model of the complex also offers a plausible structural explanation for the preference of protein kinase A for phosphorylation of Ser16 of PLN.

Animals↗
PubMed2002

Model-free analysis of protein backbone motion from residual dipolar couplings.

On the basis of the measurement of NH residual dipolar couplings (RDCs) in 11 different alignment media, an RDC-based order parameter is derived for each residue in the protein ubiquitin. Dipolar couplings are motionally averaged in the picosecond to millisecond time range and, therefore, reflect motion slower than the inverse overall tumbling correlation time of the protein. It is found that there is considerable motion that is slower than the correlation time and could not be detected with previous NMR methodology. Amplitudes and anisotropies of the motion can be derived from the model-free analysis. The method can be applied provided that at least five sufficiently different alignment media can be found for the biomolecule under investigation.

Computer Simulation↗
PubMed2002

NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein.

We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.

Amino Acid Sequence↗
PubMed2001

A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy.

The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S-hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k(1)* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k(1)* was found to be 0.02 s(-1) for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg(-1). Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg(-1). The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.

Carbon-Sulfur Ligases↗
PubMedDate not supplied

Mass spectrometric sequencing of individual peptides from combinatorial libraries via specific generation of chain-terminated sequences.

Combinatorial peptide libraries are a versatile tool for drug discovery. On-bead assays identify reactive peptides by enzyme-catalyzed staining and, usually, sequencing by Edman degradation. Unfortunately, the latter method is expensive and time-consuming and requires free N termini of the peptides. A method of rapid and unambiguous peptide sequencing by utilizing synthesis-implemented generation of termination sequences with subsequent matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis is introduced here. The required capped sequences are determined and optimized for a specific peptide library by a computer algorithm implemented in the program Biblio. A total of 99.7% of the sequences of a heptapeptide library sample could be decoded utilizing a single bead for each spectrum. To synthesize these libraries, an optimized capping approach has been introduced.

Algorithms↗
PubMedDate not supplied

In vitro generation of cytolytic T cells against human melanoma cells overexpressing HDM2.

BACKGROUND: Previous experiments have shown that tumour-associated antigens can be exploited for a successful anti-tumour immunisation. Previous reports demonstrated that oncoprotein MDM2 (HDM2) contains two highly conserved MHC class I binding motifs, MDM2100 and MDM2441, and that dendritic cells (DC) presenting MDM2100 stimulate an effective CTL reaction against melanoma cells. MATERIALS AND METHODS: In this study, we investigated the CTL-inducing capacity of autologous human dendritic cells pulsed with fragment HDM2441. RESULTS: In vitro HDM2441-primed T lymphocytes revealed a strong proliferation activity, released Th-1-associated cytokines, and possessed an effective anti-tumour activity causing apoptosis in HDM2441-overexpressing melanoma cells. Cytotoxic assay demonstrated that in parallel to melanoma cells, up to 65% of primed Tcells also underwent apoptosis. CONCLUSION: These data suggest that HDM2441 may be exploited for broad-spectrum DC-based trials against metastatic melanomas overexpressing HDM2, and point out that the efficacy of such immunotherapeutical approaches may be limited via T cell apoptosis.

Amino Acid Sequence↗