Andrew E. Bennett

Heteronuclear decoupling in rotating solids

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.

Chemical shift correlation spectroscopy in rotating solids: Radio frequency‐driven dipolar recoupling and longitudinal exchange

We present a new method of performing chemical shift correlation spectroscopy in solids with magic angle spinning (MAS). Its key feature is a longitudinal mixing period of π pulses that recouples the dipolar interaction. We discuss experimental results for triply‐13C‐labeled alanine and a theory combining MAS and π pulses.

Homonuclear radio frequency-driven recoupling in rotating solids

We discuss several aspects of homonuclear recoupling and longitudinal exchange using rotor-synchronized spin echo sequences in solid state magic-angle spinning (MAS) experiments. These include the accurate measurement of weak dipole–dipole couplings between rare spins, the behavior of dipolar trajectories in multiple spin environments, and chemical shift correlation spectroscopy via polarization exchange. To describe dipolar trajectories accurately, we adopt an approach to the simulation of these experiments which includes finite pulses and the influence of coherence decay. The latter effect becomes competitive with the strength of weak couplings in many experiments, and a simple empirical approach is outlined for the selection of decay parameters. Dipolar trajectories are shown to be dominated by the largest couplings in multiple spin systems via comparison of two and three interacting spins. Two-dimensional correlation spectroscopy based on dipolar exchange among proximate nuclei is illustrated with a u...

Recoupling of Homo- and Heteronuclear Dipolar Interactions in Rotating Solids

The measurement of homo- and heteronuclear dipolar couplings by nuclear magnetic resonance (NMR) techniques is an important tool for the determination of molecular structure in solids. In a static polycrystalline solid, the dipolar coupling between two magnetically dilute spins results in the characteristic “Pake pattern” [1], first observed in the 1H spectrum of gypsum, CaSo4-2H2O, which arises from the interaction between the two protons in the water molecules of hydration. The splitting between the singularities provides a straightforward measurement of the dipolar coupling constant and therefore the internuclear distance between the two spins. Unfortunately, in the more general case, the structural information revealed by internuclear distances cannot be obtained directly from the static 1H NMR spectrum because of the multiplicity of couplings. In situations involving other nuclei, such as 13C, 15N, and 31P, large chemical shift anisotropics, as well as other line-broadening mechanisms, obscure the lineshape perturbations from the through-space dipolar couplings.

Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation–thioesterase (T–TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4′-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T–TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T–TE interaction described here provides a model for NRPS, PKS and FAS function in general as T–TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.

FREQUENCY-SELECTIVE HETERONUCLEAR RECOUPLING IN ROTATING SOLIDS

In solid state nuclear magnetic resonance spectroscopy, several methods have recently been introduced for the purpose of restoring dipolar couplings into magic angle spinning experiments. These powerful techniques are useful for the measurement of internuclear distances and, more generally, for filtering and correlation experiments in noncrystalline materials. In the case of heteronuclear spins, the rotational echo double resonance experiment provides an elegant and practical approach to the spectrally nonselective reintroduction of dipolar interactions. In this article, we describe an approach to restore heteronuclear couplings which is spectrally selective—in particular we recouple the observed spins only with those nonobserved spins lying at exact resonance (and at multiples of the spinning frequency) in the triple resonance experiment. We refer to the technique as frequency‐selective dipolar recoupling.

Homonuclear correlation spectroscopy in rotating solids

Abstract A two-dimensional homonuclear correlation experiment for magic angle spinning NMR spectroscopy of polycrystalline solids is described. The approach involves application of a multiple pulse mixing period that scales chemical shifts to zero, thereby removing the barrier to spin exchange among interacting nuclei. Transfer of spin coherence is achieved by a transverse mixing sequence in which eight π pulses, πx-πx-πy-πy-π−y-π−y-π−x-π−x, are applied per rotor period. Spectra of triply-labeled 1,2,3-13C3-D,L-alanine are presented, which demonstrate correlations among all three carbon spins.

Frequency‐selective heteronuclear dephasing by dipole couplings in spinning and static solids

A compensated pulse sequence for the spectrally selective reintroduction of heteronuclear dipole–dipole interactions (frequency‐selective dipolar recoupling) into solid state magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments is described and shown to provide frequency‐selective dipolar dephasing in weakly coupled spin systems. The experimental dipolar spin evolution is interpreted via analytical and numerical calculations, which include a simple model for the observed losses of spin coherence in the multiple pulse experiments. In the peptide glycylglycine, the selective dipolar evolution of two spins is observed while the influence of larger internuclear couplings is suppressed. This approach is aimed at obtaining several quantitative internuclear distances independently in dipolar ‘‘recoupling’’ MAS experiments by reducing multiple spin effects in the observed dipolar evolution. Similar frequency‐selective dephasing experiments are also introduced for static solids, where an efficie...

Broadband 13C-13C adiabatic mixing in solution optimized for high fields.

Experiments which require mixing among spins with large frequency differences are generally performed with sequences based on composite pulses or computer-optimized cycles. Adiabatic pulses generally offer several advantages over other approaches, including greater single spin inversion bandwidths and tolerance to RF inhomogeneity. Here, a novel theoretical framework is presented in order to understand how spin-spin interactions are influenced by adiabatic inversion pulses, and insights from this approach are used to design more efficient adiabatic coherence exchange experiments. For very large frequency differences, this new approach generally offers improved results over previously applied mixing sequences, as applied to 13C-13C experiments which are the basis of modern sidechain assignment techniques in proteins. It is also anticipated that the approach presented here will be applicable to the analysis of various alternative approaches to adiabatic mixing.