A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment

A new strategy of backbone resonance assignment is proposed based on a combination of the most sensitive TROSY-type triple resonance experiments such as TROSY-HNCA and TROSY-HNCO with a new 3D multiple-quantum HACACO experiment. The favourable relaxation properties of the multiple-quantum coherences and signal detection using the 13C′ antiphase coherences optimize the performance of the proposed experiment for application to larger proteins. In addition to the 1HN, 15N,13Cα and 13C′ chemical shifts the 3D multiple-quantum HACACO experiment provides assignment for the 1Hα resonances in constrast to previously proposed experiments for large proteins. The strategy is demonstrated with the 44kDa uniformly 15N,13C-labeled and fractionally 35% deuterated trimeric B.subtilis Chorismate Mutase measured at 20 °C and 9 °C. Measurements at the lower temperature indicate that the new strategy can be applied to even larger proteins with molecular weights up to 80kD

Backbone resonance assignment in large protonated proteins using a combination of new 3D TROSY-HN(CA)HA, 4D TROSY-HACANH and 13C-detected HACACO experiments

A new method for backbone resonance assignment suitable for large proteins with the natural 1H isotope content is proposed based on a combination of the most sensitive TROSY-type triple-resonance experiments. These techniques include TROSY-HNCO, 13C′-detected 3D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. The favorable relaxation properties of the multiple-quantum coherences, signal detection using the 13C′ antiphase coherences, and the use of TROSY optimize the performance of the proposed set of experiments for application to large protonated proteins. The method is demonstrated with the 44 kDa uniformly 15N,13C-labeled and fractionally (35%) deuterated trimeric B. Subtilis Chorismate Mutase and is suitable for proteins with large correlation times but a relatively small number of residues, such as membrane proteins embedded in micelles or oligomeric protein

A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy

The assignment of the aliphatic 13C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) 13C-start and 13C-observe NMR experiments. These experiments start with 13C excitation and end with 13C observation while relying on the long transverse relaxation times of 13C spins in uniformly deuterated and 13C,15N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce 13C T 1 relaxation times with little effect on 13C T 2 relaxation times. Such 2D 13C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain 1H spins, the transfer of the side-chain spin polarization to the 1HN spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic 13C resonances, the side-chain 1H chemical shifts can be assigned in a semiautomatic fashion using 3D 15N-resolved and 13C-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the 1H,1H NOEs in combination with multiple residual dipolar couplings between adjacent 13C spins determined with 2D 13C-only experiment

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of 15N-15N and 1H-15N trans-hydrogen bond scalar couplings, 2h J NN and 1h J HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic model

Validated determination of NRG1 Ig-like domain structure by mass spectrometry coupled with computational modeling

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure

Leading logarithmic QCD corrections to the B_s --> \gamma \gamma decays in the two Higgs doublet model

We calculate the leading logarithmic QCD corrections to the decay B_s --> \gamma \gamma in the two Higgs doublet model (2HDM) including O_7 type long distance effects and estimate the restrictions of the 2HDM parameters, tan\beta and m_H, using the experimental data of B --> X_s \gamma decay provided by the CLEO collaboration. A lower bound for the charged Higgs mass m_H as a function of the renormalization scale \mu is given for 2HDM model II. We further present the dependencies of the branching ratio Br(B_s --> \gamma \gamma) and the ratio |A^{+}|^2/|A^{-}|^2 on m_H and tan\beta including leading logarithmic QCD corrections. The dependence on the renormalization scale is found to be strong for both ratios. An additional uncertainty arises from the variation of the parameters of the bound state model, (m_b, \bar{\Lambda}_s). We see, that to look for charged Higgs effects the measurement of the branching ratio Br(B_s --> \gamma \gamma) is promising.Comment: 25 pages, 19 figures (required epsf.sty

Validated determination of NRG1 Ig-like domain structure by mass spectrometry coupled with computational modeling

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure

Study of the Process e+ e- --> omega pi0 --> pi0 pi0 gamma in c.m. Energy Range 920--1380 MeV at CMD-2

The cross section of the process e+ e- --> omega pi0 --> pi0 pi0 gamma has been measured in the c.m. energy range 920-1380 MeV with the CMD-2 detector. Its energy dependence is well described by the interference of the rho(770) and rho'(1450) mesons decaying to omega pi0. Upper limits for the cross sections of the direct processes e+ e- --> pi0 pi0 gamma, eta pi0 gamma have been set.Comment: Accepted for publication in PL

A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment

ISSN:0925-2738ISSN:1573-500