Localising individual atoms of tryptophan side chains in the metallo-<i>β</i>-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites

The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

NUScon: a community-driven platform for quantitative evaluation of nonuniform sampling in NMR

Although the concepts of nonuniform sampling (NUS​​​​​​​) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago (Bodenhausen and Ernst, 1981; Barna and Laue, 1987), it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether through detection of closely spaced peaks (i.e., “resolution”) or peaks of weak intensity (i.e., “sensitivity”). Wider adoption of these methods is the result of improvements in computational performance, a growing abundance and flexibility of software, support from NMR spectrometer vendors, and the increased data sampling demands imposed by higher magnetic fields. However, the identification of best practices still remains a significant and unmet challenge. Unlike the discrete Fourier transform, non-Fourier methods used to reconstruct spectra from NUS data are nonlinear, depend on the complexity and nature of the signals, and lack quantitative or formal theory describing their performance. Seemingly subtle algorithmic differences may lead to significant variabilities in spectral qualities and artifacts. A community-based critical assessment of NUS challenge problems has been initiated, called the “Nonuniform Sampling Contest” (NUScon), with the objective of determining best practices for processing and analyzing NUS experiments. We address this objective by constructing challenges from NMR experiments that we inject with synthetic signals, and we process these challenges using workflows submitted by the community. In the initial rounds of NUScon our aim is to establish objective criteria for evaluating the quality of spectral reconstructions. We present here a software package for performing the quantitative analyses, and we present the results from the first two rounds of NUScon. We discuss the challenges that remain and present a roadmap for continued community-driven development with the ultimate aim of providing best practices in this rapidly evolving field. The NUScon software package and all data from evaluating the challenge problems are hosted on the NMRbox platform.

Spin noise gradient echoes

Nuclear spin noise spectroscopy in the absence of radio frequency pulses was studied under the influence of pulsed field gradients (PFGs) on pure and mixed liquids. Under conditions where the radiation-damping-induced line broadening is smaller than the gradient-dependent inhomogeneous broadening, echo responses can be observed in difference spectra between experiments employing pulsed field gradient pairs of the same and opposite signs. These observed spin noise gradient echoes (SNGEs) were analyzed through a simple model to describe the effects of transient phenomena. Experiments performed on high-resolution nuclear magnetic resonance (NMR) probes demonstrate how refocused spin noise behaves and how it can be exploited to determine sample properties. In bulk liquids and their mixtures, transverse relaxation times and translational diffusion constants can be determined from SNGE spectra recorded following tailored sequences of magnetic field gradient pulses.

Selective excitation enables encoding and measurement of multiple diffusion parameters in a single experiment

Band selectivity to address specific resonances in a spectrum enables one to encode individual settings for diffusion experiments. In a single experiment, this could include different gradient strengths (enabling coverage of a larger range of diffusion constants), different diffusion delays, or different gradient directions (enabling anisotropic diffusion measurement). In this report, a selective variant of the bipolar pulsed gradient eddy current delay (BPP-LED) experiment, enabling selective encoding of three resonances, was implemented. As proof of principle, the diffusion encoding gradient amplitude was assigned a range dependent on the selected signal, thereby allowing the extraction of the diffusion coefficient for water and a tripeptide (Met-Ala-Ser) with optimal settings in a single experiment.

A cryogen-free, semi-automated apparatus for bullet-dynamic nuclear polarization with improved resolution

In dissolution-dynamic nuclear polarization, a hyperpolarized solid is dissolved with a jet of hot solvent. The solution is then transferred to a secondary magnet, where spectra can be recorded with improved sensitivity. In bullet-dynamic nuclear polarization this order is reversed. Pressurized gas is used to rapidly transfer the hyperpolarized solid to the secondary magnet, and the hyperpolarized solid is dissolved only upon arrival. A potential advantage of this approach is that it may avoid excessive dilution and the associated signal loss, in particular for small sample quantities. Previously, we have shown that liquid-state NMR spectra with polarization levels of up to 30 % may be recorded within less than 1 s after the departure of the hyperpolarized solid from the polarizing magnet. The resolution of the recorded spectra however was limited. The system consumed significant amounts of liquid helium, and substantial manual work was required in between experiments to prepare for the next shot. Here, we present a new bullet-DNP (dynamic nuclear polarization) system that addresses these limitations.

Fluorine NMR study of proline-rich sequences using fluoroprolines

Proline homopolymer motifs are found in many proteins; their peculiar conformational and dynamic properties are often directly involved in those proteins' functions. However, the dynamics of proline homopolymers is hard to study by NMR due to a lack of amide protons and small chemical shift dispersion. Exploiting the spectroscopic properties of fluorinated prolines opens interesting perspectives to address these issues. Fluorinated prolines are already widely used in protein structure engineering – they introduce conformational and dynamical biases – but their use as 19F NMR reporters of proline conformation has not yet been explored. In this work, we look at model peptides where Cγ-fluorinated prolines with opposite configurations of the chiral Cγ centre have been introduced at two positions in distinct polyproline segments. By looking at the effects of swapping these (4R)-fluoroproline and (4S)-fluoroproline within the polyproline segments, we were able to separate the intrinsic conformational properties of the polyproline sequence from the conformational alterations instilled by fluorination. We assess the fluoroproline 19F relaxation properties, and we exploit the latter in elucidating binding kinetics to the SH3 (Src homology 3) domain.

Analysis of conformational exchange processes using methyl-TROSY-based Hahn echo measurements of quadruple-quantum relaxation

Transverse nuclear spin relaxation is a sensitive probe of chemical exchange on timescales on the order of microseconds to milliseconds. Here we present an experiment for the simultaneous measurement of the relaxation rates of two quadruple-quantum transitions in 13CH3-labelled methyl groups. These coherences are protected against relaxation by intra-methyl dipolar interactions and so have unexpectedly long lifetimes within perdeuterated biomacromolecules. However, these coherences also have an order of magnitude higher sensitivity to chemical exchange broadening than lower order coherences and therefore provide ideal probes of dynamic processes. We show that analysis of the static magnetic field dependence of zero-, double- and quadruple-quantum Hahn echo relaxation rates provides a robust indication of chemical exchange and can determine the signed relative magnitudes of proton and carbon chemical shift differences between ground and excited states. We also demonstrate that this analysis can be combined with established Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion measurements, providing improved precision in parameter estimates, particularly in the determination of 1H chemical shift differences.

The relation between crystal structure and the occurrence of quantum-rotor-induced polarization

Among hyperpolarization techniques, quantum-rotor-induced polarization (QRIP), also known as the Haupt effect, is a peculiar one. It is, on the one hand, rather simple to apply by cooling and heating a sample. On the other hand, only the methyl groups of a few substances seem to allow for the effect, which strongly limits the applicability of QRIP. While it is known that a high tunnel frequency is required, the structural conditions for the effect to occur have not been exhaustively studied yet. Here we report on our efforts to heuristically recognize structural motifs in molecular crystals able to allow to produce QRIP.

Mechanisms of coherent re-arrangement for long-lived spin order

Long-lived spin order-based approaches for magnetic resonance rely on the transition between two magnetic environments of different symmetries, one governed by the magnetic field of the spectrometer and the other where this strong magnetic field is inconsequential. Research on the excitation of magnetic-symmetry transitions in nuclear spins is a scientific field that debuted in Southampton in the year 2000. We advanced in this field carrying the baggage of pre-established directions in NMR spectroscopy. We propose to reveal herein the part of discoveries that may have been obscured by our choice to only look at them through the experience of such pre-established directions at the time. The methodological developments that are emphasised herein are the mechanisms of translation between the symmetric and non-symmetric environments with respect to the main magnetic field B0. More specifically, we look again thoroughly at zero-quantum rotations in the starting blocks of long-lived state populations, magnetisation transfers between hyperpolarised heteronuclei, and protons. These pulse sequences seed subsequent magnetic mechanisms that contribute to further applications. For instance, we show how some of the introduced coherence rotations were combined with classical pulse blocks to obtain two-dimensional correlations between protons and heteronuclei. We hope the pulse sequence building blocks discussed herein will open further perspectives for magnetic resonance experiments with long-lived spin order.

Signal-to-noise ratio in diffusion-ordered spectroscopy: how good is good enough?

Diffusion-ordered NMR spectroscopy (DOSY) constructs multidimensional spectra displaying signal strength as a function of Larmor frequency and of diffusion coefficient from experimental measurements using pulsed field gradient spin or stimulated echoes. Peak positions in the diffusion domain are determined by diffusion coefficients estimated by fitting experimental data to some variant of the Stejskal–Tanner equation, with the peak widths determined by the standard error estimated in the fitting process. The accuracy and reliability of the diffusion domain in DOSY spectra are therefore determined by the uncertainties in the experimental data and thus in part by the signal-to-noise ratio of the experimental spectra measured. Here the Cramér–Rao lower bound, Monte Carlo methods, and experimental data are used to investigate the relationship between signal-to-noise ratio, experimental parameters, and diffusion domain accuracy in 2D DOSY experiments. Experimental results confirm that sources of error other than noise put an upper limit on the improvement in diffusion domain accuracy obtainable by time averaging.