Fast multidimensional NMR with a time resolution of a few seconds provides a new tool for high throughput screening and site-resolved real-time studies of kinetic molecular processes by NMR. Recently we have demonstrated the feasibility to record protein 1H-15N correlation spectra in a few seconds of acquisition time using a new SOFAST-HMQC experiment (Schanda and Brutscher (2005) J. Am. Chem. Soc. 127, 8014). Here, we investigate in detail the performance of SOFAST-HMQC to record 1H-15N and 1H-13C correlation spectra of proteins of different size and at different magnetic field strengths. Compared to standard 1H-15N correlation experiments SOFAST-HMQC provides a significant gain in sensitivity, especially for fast repetition rates. Guidelines are provided on how to set up SOFAST-HMQC experiments for a given protein sample. In addition, an alternative pulse scheme, IPAP-SOFAST-HMQC is presented that allows application on NMR spectrometers equipped with cryogenic probes, and fast measurement of one-bond 1H-13C and 1H-15N scalar and residual dipolar coupling constants.

SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds.

In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

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Perspectives on NMR in drug discovery: a technique comes of age

We demonstrate for different protein samples that three-dimensional HNCO and HNCA correlation spectra may be recorded in a few minutes acquisition time using the band-selective excitation short-transient sequences presented here This opens new perspectives for the NMR structural investigation of unstable protein samples and real-time site-resolved studies of protein kinetics

Speeding up three-dimensional protein NMR experiments to a few minutes.

A set of BEST triple-resonance experiments for time-optimized protein resonance assignment.

Thirty percent of a cell’s volume is filled with macromolecules, and protein chemistry in a crowded environment is predicted to differ from that in dilute solution. We quantified the effect of crow...

Protein crowding tunes protein stability.

The acquisition of multidimensional NMR spectra can be speeded up by a large factor by a projection-reconstruction method related to a technique used in X-ray scanners. The information from a small number of plane projections is used to recreate the full multidimensional spectrum in the familiar format. Projections at any desired angle of incidence are obtained by Fourier transformation of time-domain signals acquired when two or more evolution intervals are incremented simultaneously at different rates. The new technique relies on an established Fourier transform theorem that relates time-domain sections to frequency-domain projections. Recent developments in NMR instrumentation, such as increased resolution and sensitivity, make fast methods for data gathering much more practical for protein and RNA research. Hypercomplex Fourier transformation generates projections in symmetrically related pairs that provide two independent "views" of the spectrum. A new reconstruction algorithm is proposed, based on the inverse Radon transform. Examples are presented of three- and four-dimensional NMR spectra of nuclease A inhibitor reconstructed by this technique with significant savings in measurement time.

Projection−Reconstruction Technique for Speeding up Multidimensional NMR Spectroscopy

A procedure is described for determining the structure of a small molecule from a single NMR experiment. Several standard NMR sequences are combined so that the essential structural information is obtained in just one pass. Two-dimensional 13C−13C correlations (“INADEQUATE”), single- and multiple-bond 13C−1H correlations, and the conventional 13C spectrum are recorded in parallel, making use of separate receiver channels for acquisition of 13C and 1H signals. The natural-abundance 13C−13C correlation measurements employ a high-sensitivity cryogenically cooled probe, optimized for 13C detection. An extension of this “all-in-one” sequence with three parallel receivers permits the corresponding natural-abundance 15N spectra to be included.

Molecular Structure from a Single NMR Experiment

We show that the weak signal that remains after (13)C-detected experiments (the (13)C "afterglow") can still be measured with high sensitivity by proton detection. This is illustrated by the incorporation of two experiments, 2D (HA)CACO and 3D (HA)CA(CO)NNH, into a single pulse sequence that makes use of two receivers in parallel. In cases where the sensitivity is not limiting, such as applications to small proteins, the inclusion of the projection-reconstruction method permits the recording of both spectra in only 15 min. High-quality data sets for the 143 residue nuclease A inhibitor (2 °C, correlation time 17.5 ns) were obtained in 3 h, illustrating the utility of the method even in studies of moderately sized proteins.

Detecting the “Afterglow” of 13C NMR in Proteins Using Multiple Receivers

Parallel receiving technologies have recently crossed the boundary separating magnetic resonance imaging from nuclear magnetic resonance (NMR) spectroscopy. In the latter case they promise significant time-saving advantages, by enabling the detection of multiple spectra simultaneously rather than in series. Moreover the full compatibility of parallel receiving with all other advances of contemporary NMR spectroscopy promises to open even further synergies in terms of speed and analytical capabilities. The present study shows one such instance, whereby the combination of parallel receiving multinuclear technologies is made with gradient-based spatial encoding methods, to yield multiple multidimensional NMR spectra in a single scan. The potential of this combination is demonstrated by the parallel acquisition of 2D H–H and H–S correlation spectra involving different S nuclei (F, P), within a single transient. Besides its potential conceptual message about what is nowadays within reach of NMR spectroscopy, the ensuing two-dimensional parallel ultrafast NMR spectroscopy (2D PUFSY) experiment carries new opportunities for high-throughput analyses, chemical kinetics, and fast experiments on metastable hyperpolarized solutions. Parallel receiving is an integral component of modern magnetic resonance; particularly in imaging applications where it can lead to substantial accelerating factors by scanning separate regions in space. The advent of multiple receivers is also beginning to influence NMR spectroscopy technologies; not by providing spatial multiplexing, but rather by enabling the simultaneous acquisition of two or more signals arising from different nuclei. Following the introduction of parallel NMR spectroscopy, it was shown that one of its main advantages results from its use to collect two or more different kinds of multidimensional correlation experiments, within the time duration that would normally entail to collect a single spectrum. This is the principle of parallel acquisition NMR spectroscopy (PANSY), which eventually evolved into more sophisticated pulse sequences capable of affording all the 2D correlation spectra necessary for a complete assignment of small molecules—within the timescale of the slowest experiment in the multiple set. These “parallel acquisition NMR, all-in-one combination of experimental applications” (PANACEA) strategies, have since been extended to systems of various heteronuclei and adapted to protein liquid-state and solid-state NMR experiments. While these multiple receiver techniques have demonstrated that substantial time savings are possible, they have still conformed to the classical means of indirect frequency encoding, whereby a series of independent scans are charged with encoding in a step-wise manner the evolution of the F1 indirect spectral domain. The incremented repetitions thus required to discretely sample the indirect time domain t1 implies that, even if sufficient sensitivity is available, sampling considerations associated with the slowest of all experiments still dictate the execution of all remaining 2D acquisitions. It was recently shown that sparse sampling coupled to non-Fourier processing techniques can alleviate this constraint, and break the Nyquist criteria without sacrifices in resolution or spectral bandwidth. Herein we present an alternative—and arguably ultimate—form of compressing multiple 2D experiments, involving their parallel implementation while following the spatially encoded protocol enabling the multiplexing of all the information involved in every indirect dimension, in a single scan. The spatiotemporal encoding principles underlying the acquisition of 2D NMR spectra/images in a single scan have been described elsewhere in detail, and hence they are only briefly described and within the context of the parallelized experiments presented here. “Ultrafast” NMR spectroscopy is based on endowing different z positions within a sample, with the different degrees of chemical shift evolution that would normally be associated with differing t1 values. If implemented in a one-to-one z–t1 fashion, this spatiotemporal encoding leads to a linear spatial winding of the magnetizations/coherences [Eq. (1)],

Multiple Parallel 2D NMR Acquisitions in a Single Scan

Two-dimensional nitrogen–carbon NMR correlation spectra have been derived by a new reconstruction technique based on standard two-dimensional HMQC and HMBC spectra, and operating with natural 15N and 13C isotopic abundances. Compared with conventional three-dimensional spectroscopy in which 15N and 13C spins must be present in the same molecule, the reconstruction method offers two orders of magnitude improvement in sensitivity. Vitamin B-12 serves as an illustrative example. Copyright © 2007 John Wiley & Sons, Ltd.

Eriks Kupce

Papers

Projection−Reconstruction Technique for Speeding up Multidimensional NMR Spectroscopy

Molecular Structure from a Single NMR Experiment

Detecting the “Afterglow” of 13C NMR in Proteins Using Multiple Receivers

Multiple Parallel 2D NMR Acquisitions in a Single Scan

Natural-abundance 15N--13C correlation spectra of vitamin B-12.