MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy

Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering.

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Backbone Dynamics of a Free and a Phosphopeptide-Complexed Src Homology 2 Domain Studied by 15N NMR Relaxation

A simple two pulse phase modulation (TPPM) scheme greatly reduces the residual linewidths arising from insufficient proton decoupling power in double resonance magic angle spinning (MAS) experiments. Optimization of pulse lengths and phases in the sequence produces substantial improvements in both the resolution and sensitivity of dilute spins (e.g., 13C) over a broad range of spinning speeds at high magnetic field. The theoretical complications introduced by large homo‐ and heteronuclear interactions among the spins, as well as the amplitude modulation imposed by MAS, are explored analytically and numerically. To our knowledge, this method is the first phase‐switched sequence to exhibit improvement over continuous‐wave (cw) decoupling in a strongly coupled homogeneous spin system undergoing sample spinning.

Heteronuclear decoupling in rotating solids

Metabolic profiling, metabolomic and metabonomic studies mainly involve the multicomponent analysis of biological fluids, tissue and cell extracts using NMR spectroscopy and/or mass spectrometry (MS). We summarize the main NMR spectroscopic applications in modern metabolic research, and provide detailed protocols for biofluid (urine, serum/plasma) and tissue sample collection and preparation, including the extraction of polar and lipophilic metabolites from tissues. 1H NMR spectroscopic techniques such as standard 1D spectroscopy, relaxation-edited, diffusion-edited and 2D J-resolved pulse sequences are widely used at the analysis stage to monitor different groups of metabolites and are described here. They are often followed by more detailed statistical analysis or additional 2D NMR analysis for biomarker discovery. The standard acquisition time per sample is 4-5 min for a simple 1D spectrum, and both preparation and analysis can be automated to allow application to high-throughput screening for clinical diagnostic and toxicological studies, as well as molecular phenotyping and functional genomics.

A.J Shaka

Papers

Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients

Computer-optimized decoupling scheme for wideband applications and low-level operation

An improved sequence for broadband decoupling: WALTZ-16

Iterative schemes for bilinear operators; application to spin decoupling

Evaluation of a new broadband decoupling sequence: WALTZ-16