Preprints  

NMR study of large proteins such as SARS-CoV-2 Main Protease can be challenging when exchange broadening, multiple stable conformations, and back-exchanging the fully deuterated chain pose problems. We demonstrate that four-dimensional NMR, including an extension of 3D NOE-NOE spectroscopy, provides an effective tool for spectral analysis. In combination with X-ray coordinates, the 4D NMR data are particularly useful for extending and validating assignments and for probing structural features.

NMR studies can be of great help to understand the molecular mechanisms of nucleosome function. For solid-state NMR, nucleosomes need to be tightly packed together. We show that centrifugation of nucleosomes results in formation of gels with very high packing ratios yet without pronounced order in the packing and without formation of specific or stable inter-nucleosome contacts. This makes the approach suitable also for the study of proteins that bind weakly to the nucleosome.

Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the lysine side chain presumably contribute to the structures and functions of proteins, the dual nature of the lysinyl residue has not been fully understood yet, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain at the atomic scale. We describe herein a novel strategy to address the current situation using nuclear magnetic resonance spectroscopy.

We report data on nuclear polarization formed in photoreaction of intramolecular electron transfer from adenine to flavin that was revealed several years ago by R. Kaptein by CIDNP. The question whether one or two types of biradicals are formed stays unresolved so far. This work clarified the problem and confirmed that single common emissive maximum in magnetic field dependence of CIDNP may originate from single level crossing of short-lived biradical and that FAD molecule has a compact shape.

An approach is described for formulating the kinetic master equations of the time evolution of hyperpolarized NMR signals in reacting (bio)chemical systems. We take a stepwise approach to formulate mathematical models of enzyme systems that agree with standard descriptions of (bio)chemical kinetics while remaining capable of describing the time evolution of magnetization described by the Bloch-McConnell equations.

This work presents a setup for the investigation of electrochemical processes during operation (in operando) using nuclear magnetic resonance (NMR) spectroscopy. The setup was designed to minimize the interferences between the NMR instrument and the electrochemical equipment. Employing this setup, the dynamic equilibrium of carbon dioxide in aqueous bicarbonate electrolyte has been monitored in operando, revealing that it may be affected by the electrochemical equipment itself.

The folding, refolding and misfolding of biomacromolecules including proteins, DNA and RNA is an important area of biophysical research to understand functional and disease states of a cell. NMR spectroscopy provides detailed insight, both with high time and atomic resolution. These experiments put stringent requirements on signal-to-noise for often irreversible folding reactions. The review describes methodological approaches and highlights key applications.

The overall aim of the study is to provide a basis from which to improve the ability of tamoxifen family drugs to reduce the activity of a secondary target protein, calmodulin, during tumour development. The main conclusion is that the binding of a tamoxifen analogue is quite unlike that of other anti-calmodulin compounds, in that two drug molecules bring the two domains of calmodulin into close proximity, but they are not fixed in orientation relative to the protein.

Typically, low-field Overhauser Dynamic Nuclear Polarization (ODNP) experiments are 1D NMR experiments to study hydration dynamics. Here, we demonstrate the application of ODNP-enhanced 2D J-resolved (JRES) spectroscopy to improve spectral resolution beyond the limit imposed by the line broadening introduced by the paramagnetic polarizing agent. Crucial to these experiments is interleaved spectral referencing to compensate for temperature induced field drifts over the course of the experiment.

Dynamic nuclear polarization (DNP) magic angle spinning NMR yields huge signal enhancements and is attractive for signal-limited cases. We studied polarization transfer pathways underlying sensitivity and resolution in DNP experiments on tubular assemblies of HIV-1 CA protein. All three polarization transfer pathways are simultaneously active and can be selected by carefully choosing experimental conditions. Our study highlights the unique capabilities of DNP for structural biology applications.

We present a system for facilitated sample vitrification, melting, and transfer in dissolution dynamic nuclear polarization experiments. The system enables inserting a sample through an airlock in combination with a dedicated dissolution system that is inserted through the same airlock shortly before the melting event. The cryostat can thus be operated continuously at low temperatures and the melting process is rapid as no pressurization steps of the cryostat are necessary for dissolution.

DEER is a method, which provide information on the structure of proteins by attaching to the protein two spins labels at well-defined location and measuring the distance between them. The sensitivity of the method in terms of the amount of the protein that is needed for the experiment depends strongly on the relaxation properties of the spin label and the composition of the solvent. In this work we show how to set up the experiment for best sensitivity when the solvent is water, H₂O.

Trigger factor (TF) is a conserved bacterial molecular chaperone that exists in a monomer-dimer equilibrium in solution. It binds to the ribosome as a monomer to facilitate folding of nascent polypeptide chains. We showed that dimeric TF exhibits distinct domain dynamics and conformational polymorphism, and that TF contains multiple substrate binding sites that are only accessible in its monomeric form. The equilibrium of TF in different oligomeric states may serve as an regulatory mechanism.

The NMR-MOUSE is a magnetic resonance tool for non-destructive materials testing inside a laboratory. The history and use of this sensor are reviewed with attention to issues encountered when employed outside. Improvements are outlined to facilitate outdoor measurements.

The iron-sulfur cluster binding properties of human mitoNEET have been investigated by 1D and 2D ¹H paramagnetic NMR spectroscopy. The NMR spectra of both oxidized and reduced mitoNEET are significantly different from those reported previously for others [Fe₂S₂] proteins. Our findings revealed the unique electronic properties of mitoNEET and suggests that the specific electronic structure of the cluster possibly drives the functional properties of different [Fe₂S₂] proteins.

Derivatives of the amino acids tryptophan and tyrosine were studied in their potential to enhance their signal in nuclear magnetic resonance spectroscopy (NMR) by laser-induced dynamic nuclear polarisation (CIDNP). Signal enhancement up to two orders of magnitude was obtained.

Catalytic hydrogenation of unsaturated molecular precursors with parahydrogen provides a major signal enhancement in NMR, which is vital for many technical as well as biomedical applications. We show that dilution of catalytically active Pd with a less active metal in heterogeneous Pd-based catalysts provides significantly larger enhancements, and that the judicious choice of the dilution metal can further improve the performance, as revealed by comparing Pd-Ag and Pd-In bimetallic catalysts.

In this work we explore how a magnetic resonance information can be obtained from simple field dependencies of reaction yields for recombining radical pairs, without actually driving any resonance transitions. An ESR spectrum can be observed in the region of relatively weak, but nonzero, applied field due to interference in the coherently created spin system of the radical pair. We also discuss connections between the level (anti)crossing and conventional magnetic resonance spectroscopy.

Shaped radiofrequency pulses are widely used in NMR spectroscopy to provide selective excitation or inversion of magnetization. Efficient accurate methods for calculating the performance of such pulses enable understanding of existing pulses and facilitate optimization of new pulses. A new approach for approximating the effects of shaped pulses is introduced and applied to some popular shaped pulses as examples. This approach also will be useful in other areas of theoretical NMR spectroscopy.

We show here that information about protein molecular dynamics can be directly extracted out of a single NMR spectrum. Understanding protein dynamics is essential to understanding its function, energetics and biology. We hope that this contribution helps bring this insight to a much broader application field and audience community than previously possible.

While most proteins adopt a single well-folded structure, several are known which interconvert among two or more conformations. Here we examine one system which is in equilibrium between two folds differing by a +3 shift in beta-strand register, describing sequence and environmental factors that bias the populations of the two structures. Notably, small molecule binding shifts the equilibrium, setting ways to artificially control natural or engineered proteins with this domain.

The application of anisotropic NMR parameters for the correct structural assignment of small molecules requires the use of partially ordered media. Previously we demonstrated that P3D simulations allows the determination of the correct diastereomer. Here we show that when steric or electrostatic factors dominate the alignment, P3D retains its diastereomer discrimination power and can define conformational ensembles of flexible molecules from sparse NMR data.

The demonstration of the Finite Pulse Radio Frequency Driven Recoupling experiment was firstly published over the past 20 years. Since it has become one of the used experiments. In this article we consider that experiment using other theoretical and simulated approaches with respect to the previous works. These approaches help us to understand more clearly the influence of each part of that experiment and to see how exactly the spin system is evaluated during it.

We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. We asked whether a detailed analysis of the individual stability curves could provide additional site-specific information. By monitoring both cold and heat denaturation of individual residues we convincingly demonstrate that the two unfolding processes are intrinsically different.

Nowadays, almost all MRI technique used in clinical applications are based on a Fourier transformation of the raw data. Alternative non-Fourier magnetic resonance imaging techniques can offer some advantages under certain conditions. SPatio-temporal ENcoding (SPEN) is a non-Fourier method that usually employs frequency-swept radio frequency pulses and can give good images in inhomogeneous magnetic fields. In this work we show how one can improve this technique by using quadratic gradients.