Real‐Time NMR Spectroscopy for Studying Metabolism

Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in‐cell NMR spectroscopy experiments. We are able to monitor real‐time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer‐based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.     

In‐Cell NMR and EPR Spectroscopy of Biomacromolecules

The dream of cell biologists is to be able to watch biological macromolecules perform their duties in the intracellular environment of live cells. Ideally, the observation of both the location and the conformation of these macromolecules with biophysical techniques is desired. The development of many fluorescence techniques, including superresolution fluorescence microscopy, has significantly enhanced our ability to spot proteins and other molecules in the crowded cellular environment. However, the observation of their structure and conformational changes while they attend their business is still very challenging. In principle, NMR and EPR spectroscopy can be used to investigate the conformation and dynamics of biological macromolecules in living cells. The development of in‐cell magnetic resonance techniques has demonstrated the feasibility of this approach. Herein we review the different techniques with a focus on liquid‐state in‐cell NMR spectroscopy, provide an overview of applications, and discuss the challenges that lie ahead.     

Probing the Intracellular Glutathione Redox Potential by In‐Cell NMR Spectroscopy

Non‐invasive and real‐time analysis of cellular redox processes has been greatly hampered by lack of suitable measurement techniques. Here we describe an in‐cell nuclear magnetic resonance (NMR) based method for measuring the intracellular glutathione redox potential by direct and quantitative measurement of isotopically labeled glutathione introduced exogenously into living yeast. By using this approach, perturbations in the cellular glutathione redox homeostasis were also monitored as yeast cells were subjected to oxidative stress.     

Single‐Scan 2D Hadamard NMR Spectroscopy

Scan and deliver : By combining imaging‐based spectral/spatial 2D radiofrequency manipulations (see scheme, left) with Hadamard‐weighting principles, 2D NMR spectra can be retrieved within a single scan (right). This approach can give homo‐ or heteronuclear correlations with an enhanced sensitivity over conventional ultrafast 2D NMR spectroscopy.

     

Real‐time mechanistic monitoring of an acetal hydrolysis using ultrafast 2D NMR

Ultrafast (UF) 2D NMR makes it possible to obtain a 2D NMR spectrum in less than a second. Here, UF‐HSQC experiments are used for the real‐time mechanistic study of an acetal hydrolysis at ¹³C natural abundance, and it is possible to characterize the presence of the hemiacetal, an intermediate with a well‐known short lifetime. The assignments are confirmed and rationalized by quantum calculations of ¹H and ¹³C NMR chemical shifts and natural bonding orbital analysis. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative Analysis of Location‐ and Sequence‐Dependent Deamination by APOBEC3G Using Real‐Time NMR Spectroscopy

The human antiretroviral factor APOBEC3G (A3G) deaminates the newly synthesized minus strand of the human immunodeficiency virus 1 (HIV‐1), which results in the abolition of the infectivity of virus‐infectivity‐factor (Vif)‐deficient HIV‐1 strains. 1 – 6 A unique property of A3G is that it deaminates a CCC hot spot that is located close to the 5′ end more effectively than one that is less close to the 5′ end. However, the mechanism of this process is elusive as it includes nonspecific binding of A3G to DNA and sliding of A3G along the DNA strand. Therefore, this process cannot be analyzed by existing methods using the Michaelis–Menten theory. A new real‐time NMR method has been developed to examine the nonspecific binding and the sliding processes explicitly, and it was applied to the analysis of the deamination by A3G. As a result, the location‐dependent deamination can be explained by a difference in the catalytic rates that depend on the direction of the approach of A3G to the target cytidine. Real‐time NMR experiments also showed that A3G deaminates CCCC tandem hotspots with little redundancy, which suggests that A3G efficiently mutates many CCC hotspots that are scattered throughout the HIV‐1 genome.     

Real‐Time Monitoring of New Delhi Metallo‐β‐Lactamase Activity in Living Bacterial Cells by 1H NMR Spectroscopy

Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D ¹H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo‐β‐lactamase subclass 1 (NDM‐1), an emerging antibiotic‐resistance threat. Cell‐based NMR studies demonstrated that two known NDM‐1 inhibitors, L ‐captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM‐1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM‐1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole‐cell settings, including mammalian cells.     

Uniform Reduction of Scalar Coupling by Real‐Time Homonuclear J‐Downscaled NMR

Scalar coupling in proton NMR spectra provides important information for the structural analysis. However, the low resolution due to the resulting signal splitting, together with the rather narrow spectral range of hydrogen, often prevents the extraction of J‐coupling information. Here we present a method to achieve real‐time homonuclear J‐downscaling. Thereby, all J‐values are uniformly reduced by an arbitrary scaling factor. In the resulting one‐dimensional spectra, signal overlap is reduced, while scalar coupling information is still available.     

Progress in Hyperpolarized Ultrafast 2D NMR Spectroscopy

An important development in the field of NMR spectroscopy has been the advent of hyperpolarization approaches, capable of yielding nuclear spin states whose value exceeds by orders‐of‐magnitude what even the highest‐field spectrometers can afford under Boltzmann equilibrium. Included among these methods is an ex situ dynamic nuclear polarization (DNP) approach, which yields liquid‐phase samples possessing spin polarizations of up to 50 %. Although capable of providing an NMR sensitivity equivalent to the averaging of about 1 000 000 scans, this methodology is constrained to extract its “superspectrum” within a single—or at most a few—transients. This makes it a poor starting point for conventional 2D NMR acquisition experiments, which require a large number of scans that are identical to one another except for the increment of a certain t₁ delay. It has been recently suggested that by merging this ex situ DNP approach with spatially encoded “ultrafast” methods, a suitable starting point could arise for the acquisition of 2D spectra on hyperpolarized liquids. Herein, we describe the experimental principles, potential features, and current limitations of such integration between the two methodologies. For a variety of small molecules, these new hyperpolarized ultrafast experiments can, for equivalent overall durations, provide heteronuclear correlation spectra at significantly lower concentrations than those currently achievable by conventional 2D NMR acquisitions. A variety of challenges still remain to be solved before bringing the full potential of this new integrated 2D NMR approach to fruition; these outstanding issues are discussed.     

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

¹H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution ¹H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.     

Real‐Time Band‐Selective Homonuclear Proton Decoupling for Improving Sensitivity and Resolution in Phase‐Sensitive J‐Resolved Spectroscopy

Real‐time band‐selective homonuclear ¹H decoupling during data acquisition of z‐filtered J‐resolved spectroscopy produces ¹H‐decoupled ¹H NMR spectra and leads to sensitivity enhancement and improved resolution, and thus aids the measurement of J couplings and residual dipolar couplings in crowded regions of ¹H NMR spectrum. High quality spectra from peptides, organic molecules, and also from enantiomers dissolved in weakly aligned chiral media are reported.     

Accelerating Diffusion‐Ordered NMR Spectroscopy by Joint Sparse Sampling of Diffusion and Time Dimensions

Diffusion‐ordered multidimensional NMR spectroscopy is a valuable technique for the analysis of complex chemical mixtures. However, this method is very time‐consuming because of the costly sampling of a multidimensional signal. Various sparse sampling techniques have been proposed to accelerate such measurements, but they have always been limited to frequency dimensions of NMR spectra. It is now revealed how sparse sampling can be extended to diffusion dimensions.     

Real‐Time Probing of Ion Pairing Dynamics with 2DIR Spectroscopy

Chemical exchange two‐dimensional infrared (2DIR) spectroscopy is applied to investigate ion pairing dynamics occurring on picosecond timescales. SeCN^? ion is used as a vibrational probe. The SeCN^? ion dissolved in N,N‐dimethyl formamide (DMF) has a sufficiently long vibrational lifetime and can form a contact ion pair with Li⁺ ion in DMF. The CN stretch frequency of the contact ion pair is significantly blue‐shifted from that of free SeCN^? so the free SeCN^? ion can be spectrally distinct from the contact ion pair in DMF. Therefore, we were able to directly monitor the ion pairing dynamics of Li⁺ and SeCN^? in real time by using ultrafast 2DIR spectroscopy. As a result, we have determined the dissociation time constant of the LiSeCN contact ion pair to be 420±40 ps.     

Laboratory Real‐Time and In Situ Monitoring of Mechanochemical Milling Reactions by Raman Spectroscopy

Mechanistic understanding of mechanochemical reactions is sparse and has been acquired mostly by stepwise ex situ analysis. We describe herein an unprecedented laboratory technique to monitor the course of mechanochemical transformations at the molecular level in situ and in real time by using Raman spectroscopy. The technique, in which translucent milling vessels are used that enable the collection of a Raman scattering signal from the sample as it is being milled, was validated on mechanochemical reactions to form coordination polymers and organic cocrystals. The technique enabled the assessment of the reaction dynamics and course under different reaction conditions as well as, for the first time, direct insight into the behavior of liquid additives during liquid‐assisted grinding.