Showing papers by "Christian Griesinger published in 2000"

A new approach for applying residual dipolar couplings as restraints in structure elucidation.

Abstract: Residual dipolar couplings are useful global structural restraints. The dipolar couplings define the orientation of a vector with respect to the alignment tensor. Although the size of the alignment tensor can be derived from the distribution of the experimental dipolar couplings, its orientation with respect to the coordinate system of the molecule is unknown at the beginning of structure determination. This causes convergence problems in the simulated annealing process. We therefore propose a protocol that translates dipolar couplings into intervector projection angles, which are independent of the orientation of the alignment tensor with respect to the molecule. These restraints can be used during the whole simulated annealing protocol.

DipoCoup: A versatile program for 3D-structure homology comparison based on residual dipolar couplings and pseudocontact shifts.

Abstract: A program, DipoCoup, is presented that allows to search the protein data bank for proteins which have a three dimensional fold that is at least partially homologous to a protein under investigation. The three dimensional homology search uses secondary structure alignment based on chemical shifts and dipolar couplings or pseudocontact shifts for the three dimensional orientation of secondary structure elements. Moreover, the program offers additional tools for handling and analyzing dipolar couplings.

A new method for the simultaneous measurement of magnitude and sign of 1DCH and 1DHH dipolar couplings in methylene groups.

Abstract: Heteronuclear dipolar couplings of the protein backbone have proven to have a big impact on the accuracy of protein NMR structures. H,H dipolar couplings might have the same impact on side chains. Here we present a method that combines both heteronuclear and homonuclear dipolar couplings to investigate the local conformation of methylene groups. A new pulse sequence (SPITZE-HSQC) is presented, that allows to measure the two C,H and the H,H dipolar couplings at the same time, using spin state selective transfers. The new method has been applied to the methylene groups of glycines in the protein ubiquitin. The C,H and the H,H dipolar couplings might have a key role in fast stereospecific assignment of protons in CH2 groups.

Adiabatic TOCSY for C,C and H,H J-transfer.

Abstract: Adiabatic pulses have been widely used for broadband decoupling and spin inversion at high magnetic fields. In this paper we propose adiabatic pulses and supercycles that can be used at high magnetic fields like 800 or 900 MHz to obtain broadband TOCSY sequences with C,C or H,H J-transfer. The new mixing sequences are equal or even superior to the well known DIPSI-2,3 experiments with respect to bandwidth. They prove robust against pulse miscalibration and B1 inhomogeneity and are therefore attractive for fully automated spectrometer environments. These adiabatic mixing sequences have been incorporated in a novel z-filter HCCH-TOCSY experiment.

Determination of ψ Torsion Angle Restraints from 3J(Cα,Cα) and 3J(HN,Cα) Coupling Constants in Proteins.

Abstract: Homonuclear 3J(Cα,Cα) and heteronuclear 3J(Cα,HN) coupling constants have been determined in the protein ubiquitin. Despite the fact that all amide bonds in ubiquitin have a trans conformation, considerable spread in the size of the coupling constants can be observed. The 3J(Cα,HN) coupling constants vary from 0.0 to 1.0 Hz, and the 3J(Cα,Cα) coupling constants that could be determined vary from 1.1 to 2.2 Hz. Interpretation of the coupling constants reveals a non-Karplus-type dependence and suggests that vicinal homonuclear 3J(Cα,Cα) and heteronuclear 3J(Cα,HN) depend on the ψi-1 torsion angle. The proposed sensitive E.COSY-type HNCO[Cα] experiment for the measurement of vicinal 3J(Cα,HN) coupling constants can be used in protonated and deuterated proteins, and the quantitative J correlation experiment HN(COCA)CA can be carried out on perdeuterated proteins for the measurement of 3J(Cα,Cα) that provide unique torsion angle information in these proton sparse proteins.

Ubiquitin Backbone Motion Studied via NHN−C‘Cα Dipolar−Dipolar and C‘−C‘Cα/NHN CSA−Dipolar Cross-Correlated Relaxation

Abstract: While it is recognized that protein side chains undergo large reorientations, very little is known about the extent and the nature of the motions within the protein backbone. These motions can be studied by chemical shift anisotropy (CSA)−dipolar cross-correlated relaxation rates, the interpretation of which relies on prior knowledge of the CSA principal axis values and directions. Alternatively, dipolar−dipolar cross-correlated relaxation rates can be used. Here we propose a CT-HNCO experiment to measure the sum of the two C‘Cα/NCα dipolar−NHN/C‘HN dipolar cross-correlated relaxation rates. At the same time the experiment gives access to the cross-correlated relaxation rates between C‘ CSA and the NHN or C‘Cα dipoles. The complete set of three cross-correlated relaxation rates, measured for the protein ubiquitin, is interpreted in terms of the Gaussian axial fluctuation model of motion. This model provides a good framework for the description of the motions of peptide planes in α-helical and turn regions...

Structure Determination of a Key Intermediate of the Enantioselective Pd Complex Catalyzed Allylic Substitution Reaction

Abstract: The structure of a catalytic intermediate with important implications for the interpretation of the stereochemical outcome of the palladium complex catalyzed allylic substitution with phosphino–oxazoline (PHOX) ligands is determined by liquid state NMR. The complex displays a novel structure that is highly distorted compared with other palladium η2-olefin complexes known so far. The structure has been determined from nuclear overhauser data (NOE), scalar coupling constants, and long range projection angle restraints derived from dipole dipole cross-correlated relaxation of multiple quantum coherence. The latter restraints have been implemented into a distance geometry protocol. The projection angle restraints yield a higher precision in the determination of the relative orientation of the two molecular moieties and are essential to provide an exact structural definition of the olefinic part of the catalytic intermediate with respect to the ligand.

N-carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea. Thermodynamics and kinetics of the spontaneous reaction.

Abstract: N-Carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v1) and cleavage (v2) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k1* of carbamate formation from ‘MFR’(MFR + MFRH+) and ‘CO2’ (CO2 + H2CO3 + HCO3−+ CO32–) was found to be 7 m−1·s−1 (v1 = k1*[‘MFR’][‘CO2’]) while the pseudo first order rate constant k2* of carbamate cleavage was 12 s−1 (v2 = k2*[carbamate]). The equilibrium constant K* = k1*/k2* = [carbamate]/[‘MFR’][‘CO2’] was 0.6 m−1 at pH 7.0 corresponding to a free energy change ΔG°′ of + 1.3 kJ·mol−1. The pH and temperature dependence of k1*, of k2* and of K* were determined. From the second order rate constant k1* it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO2 formation from HCO3− in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3− which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N-carboxymethanofuran to N-formylmethanofuran. From the concentrations of ‘CO2’ and of ‘MFR’ determined by 1D-NMR spectroscopy and the pKa of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k1 (v1 = k1[MFR][CO2]). The second order rate constant k1 was found to be approximately 1000 m−1·s−1 at 300 K and pH values between 7.0 and 8.0 which is in the order of k1 values determined for other carbamate forming reactions by stopped flow.

Determination of the Angles α and ζ in RNA by cross correlated dipolar and CSA-relaxation.

Abstract: A new method is introduced to measure the backbone torsion angles α and ζ in 13C-labeled oligonucleotides. The experiments relies on the quantification of the cross-correlated relaxation of C,P double and zero quantum coherence caused by the C,H dipolar coupling and the P chemical shift anisotropy. Two-dimensional surfaces that reveal the angular dependence of the cross-correlated relaxation rates depend on the backbone angles α and β as well as e and ζ and are interpreted using torsion angle information for the angles β and e from experiments measuring 3J(H,P) and 3J(C,P) coupling constants. The experiments have been carried out on the 10mer RNA 5‘-CGCUUUUGCG-3‘ that forms a hairpin and in which the four uridine residues are 13C-labeled in the ribofuranoside moiety.