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

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

An Improved Broadband Decoupling Sequence for Liquid Crystals and Solids

Sensitivity-enhanced two-dimensional heteronuclear shift correlation NMR spectroscopy

An improved sequence for broadband decoupling: WALTZ-16

Broadband heteronuclear decoupling

Assignment of carbon-13 NMR spectra via double-quantum coherence

Une nouvelle methode de decouplage en large bande basee sur l'inversion repetee, des spins protoniques par une sequence d'impulsions rf composees concue pour etre relativement insensibles aux effets de compensation de resonance et ete decrite anterieurement. Les auteurs developpent ici la theorie de cette methode et illustrent comment les cycles d'impulsions optimaux sont construit et comparent la theorie et l'experience

Thomas Frenkiel

Papers

An improved sequence for broadband decoupling: WALTZ-16

Broadband heteronuclear decoupling

Assignment of carbon-13 NMR spectra via double-quantum coherence

Broadband Decoupling in High-Resolution Nuclear Magnetic Resonance Spectroscopy

Supercycles for broadband heteronuclear decoupling