Gaël De Paëpe

Proton assisted recoupling and protein structure determination.

We introduce a homonuclear version of third spin assisted recoupling, a second-order mechanism that can be used for polarization transfer between (13)C or (15)N spins in magic angle spinning (MAS) NMR experiments, particularly at high spinning frequencies employed in contemporary high field MAS experiments. The resulting sequence, which we refer to as proton assisted recoupling (PAR), relies on a cross-term between (1)H-(13)C (or (1)H-(15)N) couplings to mediate zero quantum (13)C-(13)C (or (15)N-(15)N recoupling). In particular, using average Hamiltonian theory we derive an effective Hamiltonian for PAR and show that the transfer is mediated by trilinear terms of the form C(1) (+/-)C(2) (-/+)H(Z) for (13)C-(13)C recoupling experiments (or N(1) (+/-)N(2) (-/+)H(Z) for (15)N-(15)N). We use analytical and numerical simulations to explain the structure of the PAR optimization maps and to delineate the PAR matching conditions. We also detail the PAR polarization transfer dependence with respect to the local molecular geometry and explain the observed reduction in dipolar truncation. Finally, we demonstrate the utility of PAR in structural studies of proteins with (13)C-(13)C spectra of uniformly (13)C, (15)N labeled microcrystalline Crh, a 85 amino acid model protein that forms a domain swapped dimer (MW=2 x 10.4 kDa). The spectra, which were acquired at high MAS frequencies (omega(r)2pi>20 kHz) and magnetic fields (750-900 MHz (1)H frequencies) using moderate rf fields, exhibit numerous cross peaks corresponding to long (up to 6-7 A) (13)C-(13)C distances which are particularly useful in protein structure determination. Using results from PAR spectra we calculate the structure of the Crh protein.

High-resolution solid-state NMR structure of a 17.6 kDa protein.

The use of pseudocontact shifts arising from paramagnetic metal ions in a microcrystalline protein sample is proposed as a strategy to obtain unambiguous signal assignments in solid-state NMR spectra enabling distance extraction for protein structure calculation. With this strategy, 777 unambiguous (281 sequential, 217 medium-range, and 279 long-range) distance restraints could be obtained from PDSD, DARR, CHHC, and the recently introduced PAR and PAIN-CP solid-state experiments for the cobalt(II)-substituted catalytic domain of matrix metalloproteinase 12 (159 amino acids, 17.6 kDa). The obtained structure is a high resolution one, with backbone rmsd of 1.0 +/- 0.2 A, and is in good agreement with the X-ray structure (rmsd to X-ray 1.3 A). The proposed strategy, which may be generalized for nonmetalloproteins with the use of paramagnetic tags, represents a significant step ahead in protein structure determination using solid-state NMR.

Enhanced solid-state NMR correlation spectroscopy of quadrupolar nuclei using dynamic nuclear polarization.

By means of a true sensitivity enhancement for a solid-state NMR spectroscopy (SSNMR) experiment performed under dynamic nuclear polarization (DNP) conditions, corresponding to 4-5 orders of magnitude of time savings compared with a conventional SSNMR experiment, it is shown that it is possible to record interface-selective (27)Al-(27)Al two-dimensional dipolar correlation spectra on mesoporous alumina, an advanced material with potential industrial applications. The low efficiency of cross-polarization and dipolar recoupling for quadrupolar nuclei is completely negated using this technique. The important presence of pentacoordinated Al has not only been observed, but its role in bridging interfacial tetra- and hexacoordinated Al has been determined. Such structural information, collected at low temperature (∼103 K) and 9.4 T with the use of DNP, would have been impossible to obtain under standard conditions, even using a higher magnetic field. However, here it is demonstrated that this information can be obtained in only 4 h. This work clearly opens a new avenue for the application of SSNMR to quadrupolar nuclei and notably the atomic-scale structure determination of catalysis materials such as mesoporous alumina.

Solid-state NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool.

Untangling the Condensation Network of Organosiloxanes on Nanoparticles using 2D 29Si–29Si Solid-State NMR Enhanced by Dynamic Nuclear Polarization

Silica (SiO2) nanoparticles (NPs) were functionalized by silanization to produce a surface covered with organosiloxanes. Information about the surface coverage and the nature, if any, of organosiloxane polymerization, whether parallel or perpendicular to the surface, is highly desired. To this extent, two-dimensional homonuclear (29)Si solid-state NMR could be employed. However, owing to the sensitivity limitations associated with the low natural abundance (4.7%) of (29)Si and the difficulty and expense of isotopic labeling here, this technique would usually be deemed impracticable. Nevertheless, we show that recent developments in the field of dynamic nuclear polarization under magic angle spinning (MAS-DNP) could be used to dramatically increase the sensitivity of the NMR experiments, resulting in a timesaving factor of ∼625 compared to conventional solid-state NMR. This allowed the acquisition of previously infeasible data. Using both through-space and through-bond 2D (29)Si-(29)Si correlation experiments, it is shown that the required reaction conditions favor lateral polymerization and domain growth. Moreover, the natural abundance correlation experiments permitted the estimation of (2)J(Si-O-Si)-couplings (13.8 ± 1.4 Hz for surface silica) and interatomic distances (3.04 ± 0.08 Å for surface silica) since complications associated with many-spin systems and also sensitivity were avoided. The work detailed herein not only demonstrates the possibility of using MAS-DNP to greatly facilitate the acquisition of 2D (29)Si-(29)Si correlation spectra but also shows that this technique can be used in a routine fashion to characterize surface grafting networks and gain structural constraints, which can be related to a systems chemical and physical properties.

Is solid-state NMR enhanced by dynamic nuclear polarization?

The recent trend of high-field (~5-20 T), low-temperature (~100 K) ssNMR combined with dynamic nuclear polarization (DNP) under magic angle spinning (MAS) conditions is analyzed. A brief overview of the current theory of hyperpolarization for so-called MAS-DNP experiments is given, along with various reasons why the DNP-enhancement, the ratio of the NMR signal intensities obtained in the presence and absence of microwave irradiation suitable for hyperpolarization, should not be used alone to gauge the value of performing MAS-DNP experiments relative to conventional ssNMR. This is demonstrated through a dissection of the current conditions required for MAS-DNP with particular attention to resulting absolute sensitivities and spectral resolution. Consequently, sample preparation methods specifically avoiding the surplus of glass-forming solvents so as to improve the absolute sensitivity and resolution are discussed, as are samples that are intrinsically pertinent for MAS-DNP studies (high surface area, amorphous, and porous). Owing to their pertinence, examples of recent applications on these types of samples where chemically-relevant information has been obtained that would have been impossible without the sensitivity increases bestowed by MAS-DNP are also detailed. Additionally, a promising further implementation for MAS-DNP is exampled, whereby the sensitivity improvements shown for (correlation) spectroscopy of nuclei at low natural isotopic abundance, facilitate internuclear distance measurements, especially for long distances (absence of dipolar truncation). Finally, we give some speculative perspectives for MAS-DNP.

Proton assisted recoupling at high spinning frequencies.

We demonstrate the successful application of (13)C-(13)C proton assisted recoupling (PAR) on [U-(13)C,(15)N] N-f-MLF-OH and [U-(13)C,(15)N] protein GB1 at high magic angle spinning (MAS) frequencies (omega(r)/2pi = 65 kHz). Specifically, by combining PAR mixing with low power heteronuclear decoupling (omega(1H)/2pi approximately 16 kHz) and high spinning frequencies, we obtain high resolution 2D spectra displaying long-range (13)C-(13)C contacts from which distance estimates can be extracted. These experiments therefore demonstrate the possibility of performing high resolution structural studies in the limit of high spinning frequency and low power (1)H decoupling, a regime which optimizes the resolution of protein samples and preserves their integrity.

Improved heteronuclear decoupling schemes for solid-state magic angle spinning NMR by direct spectral optimization

It is shown that a direct spectral optimization scheme can be implemented to obtain improved heteronuclear dipolar decoupling schemes for solid-state magic-angle-spinning NMR experiments. The resulting schemes, which turn out to have a particularly simple form, are shown to be applicable over the whole range of commercially available spinning speeds (from 5 to 35 kHz), and are shown to improve on the performance of the best existing sequences.

Spin dynamics in the modulation frame: application to homonuclear recoupling in magic angle spinning solid-state NMR.

We introduce a family of solid-state NMR pulse sequences that generalizes the concept of second averaging in the modulation frame and therefore provides a new approach to perform magic angle spinning dipolar recoupling experiments. Here, we focus on two particular recoupling mechanisms-cosine modulated rotary resonance (CMpRR) and cosine modulated recoupling with isotropic chemical shift reintroduction (COMICS). The first technique, CMpRR, is based on a cosine modulation of the rf phase and yields broadband double-quantum (DQ) (13)C recoupling using >70 kHz omega(1,C)/2pi rf field for the spinning frequency omega(r)/2=10-30 kHz and (1)H Larmor frequency omega(0,H)/2pi up to 900 MHz. Importantly, for p>or=5, CMpRR recouples efficiently in the absence of (1)H decoupling. Extension to lower p values (3.5<or=p<5) and higher spinning frequencies is possible using low power (1)H irradiation (<0.25 omega(r)/2pi). This phenomenon is explained through higher order cross terms including a homonuclear third spin assisted recoupling mechanism among protons. CMpRR mitigates the heating effects of simultaneous high power (13)C recoupling and (1)H decoupling. The second technique, COMICS, involves low power (13)C irradiation that induces simultaneous recoupling of the (13)C DQ dipolar and isotropic chemical shift terms. In contrast to CMpRR, where the DQ bandwidth (approximately 30 kHz at omega(0,H)/2pi=750 MHz) covers the entire (13)C spectral width, COMICS recoupling, through the reintroduction of the isotropic chemical shift, is selective with respect to the carrier frequency, having a typical bandwidth of approximately 100 Hz. This approach is intended as a general frequency selective method circumventing dipolar truncation (supplementary to R(2) experiments). These new gamma-encoded sequences with attenuated rf requirements extend the applicability of homonuclear recoupling techniques to new regimes--high spinning and Larmor frequencies--and therefore should be of major interest for high resolution biomolecular studies.

Towards Structure Determination of Self-Assembled Peptides Using Dynamic Nuclear Polarization Enhanced Solid-State NMR Spectroscopy†

Bio-inspired self-assemblies made of peptide building blocks have great potential for nanotechnology ranging from biological and pharmaceutical applications to (opto)electronics. [1–3] With these goals, a variety of peptide nanoassemblies have been studied and designed over the last few decades. [4] Inevitably, structural studies at an atomic scale are crucial to unravel the mechanisms that drive nanoassembly formation as well as to relate these structures to their physical and chemical properties. However, structure determination at an atomic level is challenging essentially because of the difficulty associated with using X-ray crystallography on such nanoassemblies. [3] Solid-state NMR (SSNMR) spectroscopy is a powerful and promising technique for structural analysis of nanoassemblies. In principle, SSNMR spectroscopy can be used for any form of solid sample from well-ordered crystals to disordered powders. [5] Furthermore, the recent development