ABSTRACT
Metabolites play crucial roles in cellular processes, yet their diffusion in the densely packed interiors of cells remains poorly understood, compounded by conflicting reports in existing studies. Here, we employ pulsed-gradient stimulated-echo NMR and Brownian/Stokesian dynamics simulations to elucidate the behavior of nano- and subnanometer-sized tracers in crowded environments. Using Ficoll as a crowder, we observe a linear decrease in tracer diffusivity with increasing occupied volume fraction, persistingâsomewhat surprisinglyâup to volume fractions of 30-40%. While simulations suggest a linear correlation between diffusivity slowdown and particle size, experimental findings hint at a more intricate relationship, possibly influenced by Ficoll's porosity. Simulations and numerical calculations of tracer diffusivity in the E. coli cytoplasm show a nonlinear yet monotonic diffusion slowdown with particle size. We discuss our results in the context of nanoviscosity and discrepancies with existing studies.
ABSTRACT
1H NMR spectroscopy is a powerful tool for analyzing mixtures including determining the concentrations of individual components. When signals from multiple compounds overlap, this task requires computational solutions. They are typically based on peak-picking and the comparison of obtained peak lists with libraries of individual components. This can fail if peaks are not sufficiently resolved or when peak positions differ between the library and the mixture. In this paper, we present Magnetstein, a quantification algorithm rooted in the optimal transport theory that makes it robust to unexpected frequency shifts and overlapping signals. Thanks to this, Magnetstein can quantitatively analyze difficult spectra with the estimation trueness an order of magnitude higher than that of commercial tools. Furthermore, the method is easier to use than other approaches, having only two parameters with default values applicable to a broad range of experiments and requiring little to no preprocessing of the spectra.
ABSTRACT
Chemical analysis based on liquid-state nuclear magnetic resonance spectroscopy exploits numerous observables, mainly chemical shifts, relaxation rates, and internuclear coupling constants. Regarding the latter, the efficiencies of internuclear coherence transfers may be encoded in spectral peak intensities. The dependencies of these intensities on the experimental parameter that influences the transfer, for example, mixing time, are an important source of structural information. Yet, they are costly to measure and difficult to analyze. Here, we show that peak intensity build-up curves in two-dimensional total correlation spectroscopy (2D TOCSY) experiments may be quickly measured by employing nonuniform sampling and that their analysis can be effective if supported by quantum mechanical calculations. Thus, such curves can be used to form a new, third pseudodimension of the TOCSY spectrum. Similarly to the other two frequency dimensions, this one also resolves ambiguities and provides characteristic information. We show how the approach supports the analysis of a fragment of protein Tau Repeat-4 domain. Yet, its potential applications are far broader, including the analysis of complex mixtures or other polymers.
Subject(s)
Complex Mixtures , Magnetic Resonance Spectroscopy/methodsABSTRACT
The construction of an efficient conductive interface between electrodes and electroactive proteins is a major challenge in the biosensor and bioelectrochemistry fields to achieve the desired nanodevice performance. Concomitantly, metallo-organic terpyridine wires have been extensively studied for their great ability to mediate electron transfer over a long-range distance. In this study, we report a novel stepwise bottom-up approach for assembling bioelectrodes based on a genetically modified model electroactive protein, cytochrome c553 (cyt c553) and an organometallic terpyridine (TPY) molecular wire self-assembled monolayer (SAM). Efficient anchoring of the TPY derivative (TPY-PO(OH)2) onto the ITO surface was achieved by optimising solvent composition. Uniform surface coverage with the electroactive protein was achieved by binding the cyt c553 molecules via the C-terminal His6-tag to the modified TPY macromolecules containing Earth abundant metallic redox centres. Photoelectrochemical characterisation demonstrates the crucial importance of the metal redox centre for the determination of the desired electron transfer properties between cyt and the ITO electrode. Even without the cyt protein, the ITO-TPY nanosystem reported here generates photocurrents whose densities are 2-fold higher that those reported earlier for ITO electrodes functionalised with the photoactive proteins such as photosystem I in the presence of an external mediator, and 30-fold higher than that of the pristine ITO. The universal chemical platform for anchoring and nanostructuring of (photo)electroactive proteins reported in this study provides a major advancement for the construction of efficient (bio)molecular systems requiring a high degree of precise supramolecular organisation as well as efficient charge transfer between (photo)redox-active molecular components and various types of electrode materials.
Subject(s)
Biosensing Techniques , Electrochemistry , Electrodes , Electron Transport , Oxidation-ReductionABSTRACT
Monitoring of chemical reactions is best carried out using methods that sample the test object at a rate greater than the time scale of the processes taking place. The recently proposed time-resolved nonuniform sampling (TR-NUS) method allows the use of two-dimensional (2D) nuclear magnetic resonance (NMR) spectra for this purpose and provides a time resolution equivalent to that achievable using one-dimensional spectra. Herein, we show that TR-NUS acquired data eliminates 2D spectral line disturbances and enables more accurate signal integration and kinetics conclusions. The considerations are exemplified with a seemingly simple aza-Michael reaction of benzylamine and acrylamide. Surprisingly, the product identification is possible only using 2D spectra, although credible monitoring requires TR-NUS.
ABSTRACT
Benchtop NMR spectrometers have become widely available over the last decade. They are now used successfully in various branches of chemistry. Their popularity continues to grow due to their low price and almost zero running costs. However, benchtop spectrometers suffer from low resolution and sensitivity compared to the high-field spectrometers used in NMR labs for several decades. In this article we present a solution for boosting the sensitivity of benchtop NMR spectrometers in a multi-scan experiment and improving their capabilities in quantitative measurement. Our solution involves the synchronized shifting of a sample to preserve its high nuclear polarization during the measurement. We performed several experiments using different samples to confirm this improved performance: an 1H NMR experiment for 4,4-dimethoxy-2-butanone, and 13C NMR experiments for benzyl salicylate, the liquid pharmaceutical product Acerin (skin solution), and a mixture of m-anisaldehyde and (R)-(+)-limonene.
ABSTRACT
Modern nuclear magnetic resonance spectroscopy (NMR) is based on two- and higher-dimensional experiments that allow the solving of molecular structures, i.e., determine the relative positions of single atoms very precisely. However, rich chemical information comes at the price of long data acquisition times (up to several days). This problem can be alleviated by compressed sensing (CS)-a method that revolutionized many fields of technology. It is known that CS performs the most efficiently when measured objects feature a high level of compressibility, which in the case of NMR signal means that its frequency domain representation (spectrum) has a low number of significant points. However, many NMR spectroscopists are not aware of the fact that various well-known signal acquisition procedures enhance compressibility and thus should be used prior to CS reconstruction. In this study, we discuss such procedures and show to what extent they are complementary to CS approaches. We believe that the survey will be useful not only for NMR spectroscopists but also to inspire the broader signal processing community.
ABSTRACT
NMR spectroscopy is a versatile tool for studying time-dependent processes: chemical reactions, phase transitions or macromolecular structure changes. However, time-resolved NMR is usually based on the simplest among available techniques - one-dimensional spectra serving as "snapshots" of the studied process. One of the reasons is that multidimensional experiments are very time-expensive due to costly sampling of evolution time space. In this review we summarize efforts to alleviate the problem of limited applicability of multidimensional NMR in time-resolved studies. We focus on techniques based on sparse or non-uniform sampling (NUS), which lead to experimental time reduction by omitting a significant part of the data during measurement and reconstructing it mathematically, adopting certain assumptions about the spectrum. NUS spectra are faster to acquire than conventional ones and thus better suited to the role of "snapshots", but still suffer from non-stationarity of the signal i.e. amplitude and frequency variations within a dataset. We discuss in detail how these instabilities affect the spectra, and what are the optimal ways of sampling the non-stationary FID signal. Finally, we discuss related areas of NMR where serial experiments are exploited and how they can benefit from the same NUS-based approaches.
ABSTRACT
Triptycene derivatives are widely utilized in different fields of chemistry and materials sciences. Their physicochemical properties, often of pivotal importance for the rational design of triptycene-based functional materials, are influenced by noncovalent interactions between substituents mounted on the triptycene skeleton. Herein, a unique interaction between electron-rich substituents in the peri position and the silyl group located on the bridgehead sp3 -carbon is discussed on the example of 1,4-dichloro-9-(p-methoxyphenyl)-silyltriptycene (TRPCl) which exists in solution in the form of two rotamers differing by dispositions, syn or anti, of the Si-CPh (the CPh atom is from the p-methoxyphenyl group) bond against the peri-Cl atom. For the first time, substantial differences between the Si-CPh bonds in these two dispositions are identified, based on indirect experimental and direct theoretical evidence. For these two orientations, the experimental 1 J(Si,CPh ) values differ by as much as 10 percent. The differences are explained in terms of effective electron density transfer from the peri-Cl atom to the antibonding σ* orbitals of the Si-X bonds (X=H, CPh ) oriented anti to that atom. The electronic effects are revealed by an NBO analysis. Connections of these observations with the notion of blue-shifting hydrogen bonds are discussed.
ABSTRACT
Low-field benchtop nuclear magnetic resonance (BT-NMR) spectrometers with Halbach magnets are being increasingly used in science and industry as cost-efficient tools for the monitoring of chemical reactions, including hydrogenation. However, their use of low-field magnets limits both resolution and sensitivity. In this paper, we show that it is possible to alleviate these two problems through the combination of parahydrogen-induced polarization (PHIP) and fast correlation spectroscopy with time-resolved non-uniform sampling (TR-NUS). PHIP can enhance NMR signals so that substrates are easily detectable on BT-NMR spectrometers. The interleaved acquisition of one- and two-dimensional spectra with TR-NUS provides unique insight into the consecutive moments of hydrogenation reactions, with a spectral resolution unachievable in a standard approach. We illustrate the potential of the technique with two examples: the hydrogenation of ethylphenyl propiolate and the hydrogenation of a mixture of two substrates - ethylphenyl propiolate and ethyl 2-butynoate.
ABSTRACT
NMR spectroscopy, used routinely for structure elucidation, has also become a widely applied tool for process and reaction monitoring. However, the most informative of NMR methods-correlation experiments-are often useless in this kind of applications. The traditional sampling of a multidimensional FID is usually time-consuming, and thus, the reaction-monitoring toolbox was practically limited to 1D experiments (with rare exceptions, e.g., single-scan or fast-sampling experiments). Recently, the technique of time-resolved non-uniform sampling (TR-NUS) has been proposed, which allows to use standard multidimensional pulse sequences preserving the temporal resolution close to that achievable in 1D experiments. However, the method existed only as a prototype and did not allow on-the-fly processing during the reaction. In this paper, we introduce TReNDS: free, user-friendly software kit for acquisition and processing of TR-NUS data. The program works on Bruker, Agilent, and Magritek spectrometers, allowing to carry out up to four experiments with interleaved TR-NUS. The performance of the program is demonstrated on the example of enzymatic hydrolysis of sucrose.
ABSTRACT
Nuclear magnetic resonance (NMR) spectroscopy is frequently applied in quantitative chemical analysis (qNMR). It is easy to measure one-dimensional (1D) NMR spectra in a quantitative regime (with appropriately long relaxation delays and acquisition times); however, their applicability is limited in the case of complex samples with severe peak overlap. Two-dimensional (2D) NMR solves the overlap problem, but at the cost of biasing peak intensities and hence quantitativeness. This is partly due to the uneven coherence transfer between excited/detected 1H nuclei and the heteronuclei coupled to them (typically 13C). In the traditional approach, the transfer occurs via the evolution of a spin system state under the J-coupling Hamiltonian during a delay of a fixed length. The delay length is set on the basis of the predicted average coupling constant in the sample. This leads to disturbances for pairs of nuclei with coupling constants deviating from this average. Here, we present a novel approach based on non-standard processing of the data acquired in experiments, where the coherence transfer delay is co-incremented with non-uniformly sampled evolution time. This method allows us to obtain the optimal transfer for all resonances, which improves quantitativeness. We demonstrate the concept for the coherence transfer and multiplicity-edit delays in a heteronuclear single-quantum correlation experiment (HSQC).