We show that water saturation leads to deleterious losses in sensitivity of methyl signals in selectively methyl-[CH]-labeled protein samples of high molecular weight proteins dissolved in HO. These losses arise from efficient cross-relaxation between methyl protons and proximal labile protons in the protein structure. A phenomenological model for analysis of methyl intensity decay profiles that involves exchange saturation transfer of magnetization from localized proton spins of water to various labile groups in the protein structure that, in turn, efficiently cross-relax with protons of methyl groups, is described. Analysis of methyl intensity decay profiles with this model allows cross-relaxation rates (σ) between methyl and labile protons to be determined and permits identification of methyl sites in close proximity to labile groups in the protein structure.

We present a novel approach for side-chain-selective deuteration of proteins to improve H spectral resolution and to simplify side-chain signals in H-detected protein solid-state NMR (SSNMR) with a simple bio-expression method using E. coli BL21 (DE3). H-detected SSNMR using ultra-fast magic-angle spinning (MAS) at a spinning rate of 60 kHz or higher is attracting attention as a powerful method of protein structure determination. However, even with ultra-fast MAS at 100 kHz, the H line broadening due to H-H dipolar interactions cannot be eliminated, posing an obstacle to signal assignment and structure determination. To improve resolution for SSNMR-based protein structural analysis, we developed a method to selectively deuterate side-chains at a high deuteration level while maintaining the protons at the α-position. This selective labeling method is based on the transamination reaction in the amino-acid biosynthesis pathway and switching a medium from an unlabeled HO medium containing D-glucose (glucose), ammonium chloride, and amino acid mixture for rapid cell growth to a labeled HO medium containing [H, C]-glucose, N-labeled ammonium chloride, and a [H, C, N]-labeled amino-acid mixture just before the induction. With [H, C]-labeled glucose and a [H, C, N]-labeled amino-acid mixture as the carbon sources, this medium-switching method provides a simple and efficient means to express a selectively deuterated protein GB1 domain (GB1) sample, which is achieved by promoting efficient back-protonation at the α-position via the transamination reaction while retaining side-chain deuterons to a large extent. The yield of the GB1 protein was found to be enhanced by a factor of ca. 1.5 with the medium-switching method, compared with that for the expression with a traditional M9 minimal medium in HO without medium-switching. For the selectively deuterated GB1 sample, the resultant H resolution for resolved H peaks in H-detected 2D H/C correlation SSNMR at a MAS rate of 70 kHz was improved by a factor of 1.21 on average, compared with the corresponding resolution for a fully protonated, uniformly C- and N-labeled GB1 sample. Furthermore, side-chain signal assignment is facilitated by utilizing residual protons of the side chains. Our results also suggest that the side-chain deuteration level can be altered by adjusting the level of the deuterated amino-acid mixture in the expression system.

Cellulose nanocrystals (CNCs) is synthesized from alpha-cellulose by acid hydrolysis method, and formation of nanocrystallization is comprised by using various microscopic and spectroscopic techniques like PXRD, XPS, Raman, FTIR, PL, UV-Vis, DSC, TGA, DLS, SEM, TEM. Nanocrystalline cellulose shows a notably higher photoluminescence (PL) intensity than cellulose, which enhances its ability to absorb and emit visible light. This increase in PL intensity is attributed to a smaller particle size of CNCs, greater surface area, and quantum confinement effects. The higher intensity of the XPS spectrum further supports the larger surface area of CNCs. PXRD and Raman spectroscopy results show that CNCs has a higher crystallinity index than cellulose. Through deconvolution of the C CP-MAS SSNMR spectrum, we confirmed a significant reduction in the relative abundance of the amorphous region of cellulose (43.61%) to just 4.97% in CNCs. The C CP-MAS SSNMR spectrum of CNCs, at the C4, C6, C2C3C5 nuclei sites, can be fitted by two distinct lines for both amorphous and crystalline region, indicating the formation of a co-crystal from two nanocrystallites. Despite this, the principal components of the CSA (chemical shift anisotropy) tensor remain unchanged, suggesting similar electronic environments for these two nanocrystallites. The spin-lattice relaxation time and local correlation time of cellulose and CNCs are determined for chemically distinct carbon nuclei residing on D-glucopyranose units. It is noteworthy that the C spin-lattice relaxation time and C local correlation time are longer for each chemically distinct nucleus in CNCs compared to cellulose. It can be predicted by observing the NMR relaxometry data that the longer relaxation time in CNCs is due to the enhancement of crystallinity index. Hence, a correlation between the crystallinity index and nuclear spin dynamics can be established by NMR relaxometry measurements. These findings offer significant insights into the intricate structure and dynamic behavior of cellulose and nanocrystalline cellulose (CNCs), crucial for advancing biomimetic material design, which has huge applications across the pharmaceutical, textile, and cosmetics industries.

Recent advances in high power NMR relaxation dispersion experiments have significantly enhanced our ability to study fast µs timescale motions in proteins, which are crucial for understanding their biological functions. Here, we have extended the detectable time window of such fast dynamics with the development of extreme power H Carr-Purcell-Meiboom-Gill (H E-CPMG) experiments targeted at the backbone amide protons (H). Using this methodology, artifact-free relaxation dispersion profiles can be obtained up to extreme pulsing conditions with minimal setup effort using commonly used standard NMR hardware. We demonstrate the utility of ¹H E-CPMG on human ubiquitin, revealing that the previously reported peptide flip motion influences a larger region of the protein backbone than previously recognized. Additionally, we directly observed a faster dynamic process at residue T09, aligning with previously predicted pincer mode motion. These findings underscore the effectiveness of H E-CPMG in extending the temporal resolution at which biologically relevant fast protein dynamics can be studied.

Biomolecular dynamics in the microsecond-to-millisecond (µs-ms) timescale are linked to various biological functions, such as enzyme catalysis, allosteric regulation, and ligand recognition. In solution state NMR, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are commonly used to probe µs-ms timescale motions, providing detailed kinetic, thermodynamic, and mechanistic information at the atomic level. For investigating conformational dynamics in high-molecular-weight biomolecules, methyl groups serve as ideal probes due to their favorable relaxation properties, and C CPMG relaxation dispersion is widely employed for characterizing dynamics in selectively CH-labeled samples. However, conventional schemes that apply CPMG pulses with constant phase are susceptible to artifacts arising from off-resonance effects, radiofrequency (RF) field inhomogeneity and pulse imperfections. In this work we present an optimizedC single-quantum (SQ) CPMG experiment incorporating the [0013]-phase cycling scheme, and demonstrate its enhanced robustness against various adverse effects. Moreover, the optimized pulse scheme enables finer sampling of CPMG pulsing frequencies and is suited for studying systems with variable J scalar coupling constants, thereby facilitating comprehensive characterization of µs-ms timescale dynamics of biomolecules with increased precision.

The E. coli γ-clamp loader is a 200 kDa pentameric AAA + ATPase comprised of γ, δ and δ' subunits in a 3:1:1 ratio, which opens the ring shaped β-clamp homodimer and loads it onto DNA in a process essential for DNA replication. The clamp loading is initiated by ATP binding, which induces conformational changes in the clamp loader allowing it to bind and open the β-clamp. This is followed by DNA primer-template binding, ATP hydrolysis, and clamp release onto DNA. Despite a wealth of structural and functional data, dynamics and interactions of the γ-clamp loader and the β-clamp underlying elementary steps of this process remain elusive. Here we employed a "divide-and-conquer" strategy for the initial NMR characterization of the γ-clamp loader. A new protocol for the clamp loader assembly was proposed allowing selective incorporation of the isotope-labeled δ and δ' subunits for NMR studies. The nearly complete H, N and C NMR resonance assignments were obtained for the isolated modular domains of the δ and δ' subunits, which facilitated the assignments of the full-length subunits, and side-chain methyl assignments of the subunits in the context of pentameric γ-clamp loader. NMR chemical shift analysis using the random coil index approach revealed increased flexibility in the ATP, DNA, and β-clamp binding interfaces of the isolated subunits, highlighting a potential significance of conformational dynamics for the clamp loading process. The reported clamp loader assembly protocol and resonance assignments enable the detailed NMR studies of protein dynamics and mechanochemistry of the clamp loading cycle.

Cellular functions require biomolecules to transition among various conformational sub-states in the energy landscape. A mechanistic understanding of cellular functions requires quantitative knowledge of the kinetics, thermodynamics, and structural features of the biomolecules experiencing exchange between several states. High-power Relaxation Dispersion (RD) NMR experiments have proven very effective for such measurements if the exchange occurs in timescales ranging from microseconds to milliseconds. However, scanning the significantly larger kinetic window within the time limit of instrumental availability and sample stability requires careful optimization of experiments. Understanding biomolecular functions at a mechanistic level depends on fitting such experimental data to theoretical models. However, the reliability of the fit parameters depends on the measurement schemes and is sensitive to experimental noise. Here, we benchmark different measurement schemes along with theoretical models for sub-millisecond timescale exchange and determine the robustness of these models in providing information when the measurements contain noise. Our results show that kinetics can be measured reliably from such experiments. The structural features of the exchanging sub-states, encoded in the chemical shift differences between the states, can be fitted, albeit with significant uncertainties. Information about the minor states is difficult to obtain exclusively from the RD data due to large uncertainties and sensitivity to noise.

The use of H detection, made possible by very fast magic-angle spinning (MAS), has revolutionized the field of biomolecular solid-state NMR. In the past, H detection was often paired with deuteration schemes to achieve the highest possible resolution needed for protein structural characterization. However, with modern probes capable of MAS rates over 100 kHz, deuteration is no longer required, resulting in a need to measure long-range distances in fully protonated systems. In this study, we evaluate the potential of two 3D pulse sequences, (H)NCOH and (H)NCAH, to measure long-range C-H correlations in a fully protonated protein sample at a MAS rate of 105 kHz. Our results show that the (H)NCOH spectrum contains multiple sequential and structurally relevant long-range CO-H contacts for each residue, capturing H contacts up to 6 Å despite transfers to side chain protons. Conversely, the (H)NCAH spectrum yields fewer Cα-H correlations, with those present mostly from intraresidue aliphatic proton contacts. Therefore, in protonated proteins, the extensive H network leads to dipolar truncation in the Cα-H experiment, while the CO-H correlations observed are comparable to those in deuterated samples. These findings highlight the feasibility of conducting distance measurements based on long-range cross polarization, on more accessible and affordable samples, expanding the scope of proton detection for systems where deuteration and back-exchange are not possible.

The cellular environment is a complex and crowded space, with organelles, compartments and multitudes of molecules engaged in intricate networks of communication that modulate binary protein-ligand/protein interactions. As a result, it is becoming increasingly appreciated that evaluations of protein-drug binding should be carried out in the native cellular environment. Here, we present a proof-of-concept study where we measured the lifetime (1/k) of a protein-drug complex in human cells by F NMR spectroscopy using fluorinated Cyclophilin A (CypA) bound to Cyclosporine A (CsA). Harnessing the exceptional detection sensitivity of the trifluoromethyl group attached at the para position of Phe60 in CypA, high-quality 2D F-F exchange spectra were obtained in cells. Essentially identical k values were observed in cells and in vitro, suggesting that the overall impact of the cellular environment on the lifetime of tfmF60 CypA/CsA complex is minimal. Using similar approaches for quantifying protein-drug lifetimes in the native cellular environment paves the way for efficiently screening drug libraries in human cells by F NMR spectroscopy.

COLMARvista is presented as a new, highly versatile software for the easy and intuitive processing and visual inspection of 2D and pseudo-3D NMR data both for uniformly and non-uniformly sampled datasets. COLMARvista allows fully autonomous processing of spectra, including zero-filling, apodization, water suppression, Fourier transformation, and phase correction. Its full integration with DEEP Picker and Voigt Fitter programs allows the automated deconvolution and reconstruction of the experimental spectra for highly quantitative analysis, from compound concentration determination to the extraction of cross-peak specific relaxation parameters, even for signals affected by significant overlap with other peaks. COLMARvista is based on JavaScript and, hence, it is computer-architecture and operating-system independent including its advanced graphics. It runs on all recent web browsers and does not require a potentially elaborate operating-system dependent installation. COLMARvista may serve as a paradigm also for other software projects to prevent the stockpiling of once powerful legacy software that became frozen in time, thereby ensuring continuing progress of the NMR field and its software for future generations of NMR spectroscopists.

Magic angle spinning nuclear magnetic resonance (MAS NMR) is well suited for the determination of protein structure. The key structural information is obtained in the form of spectral cross peaks between spatially close nuclear spins, but assigning these cross peaks unambiguously to unique spin pairs is often a tedious task because of spectral overlap. Here, we use a seven-helical membrane protein Anabaena Sensory Rhodopsin (ASR) as a model system to demonstrate that transverse Paramagnetic Relaxation Enhancements (PRE) extracted from 2D MAS NMR spectra could be used to obtain a protein structural model. Starting with near complete assignments (93%) of ASR residues, TALOS + predicted backbone dihedral angles and secondary structure restraints in the form of backbone hydrogen bonds are combined with PRE-based restraints and used to generate a coarse model. This model is subsequently utilized as a template reference to facilitate automated assignments of highly ambiguous internuclear correlations. The template is used in an iterative cross peak assignment process and is progressively improved through the inclusion of disambiguated restraints, thereby converging to a low root-mean-square-deviation structural model. In addition to improving structure calculation conversion, the inclusion of PREs also improves packing between helices within an alpha-helical bundle.

Methyl groups are essential probes for characterising interactions and dynamics in large proteins. HN-based triple-resonance NMR experiments are often too insensitive for methyl assignments, making a NOESY-based approach an efficient strategy. Linking geminal methyl groups in leucine and valine residues is a crucial step in such NOESY-based methyl resonance assignment strategies. This link can be established unambiguously with the 3D-HMBC-HMQC experiment, introduced for large U-[C, H] LV-[CH]-labelled proteins. Here, we introduce the SOFAST variant of the 3D-HMBC-HMQC experiment which provides spectra with fewer artefacts arising from the water signal and a mean increase in signal-to-noise ratio per unit time of 16% compared to the original experiment with an optimised recovery delay.

Paramagnetic relaxation enhancement (PRE) is widely used in biomolecular NMR spectroscopy to obtain long-range distance and orientational information for intra- or intermolecular interactions. In contrast to conventional PRE measurements, which require tethering small molecules containing either a radical or paramagnetic ion to specific sites on the target protein, solvent PRE (sPRE) experiments utilize paramagnetic cosolutes to induce a delocalized PRE effect. Compounds developed as contrast agents in magnetic resonance imaging (MRI) applications typically consist of Gd chelated by a small molecule. Coordinating these Gd-containing small molecules to larger and inert scaffolds has been shown to increase the PRE-effect and produce more effective contrast agents in MRI. Inspired by their use as MRI contrast agent, in this work we evaluate the effectiveness of using a functionalized polyamidoamine (PAMAM) dendrimer for sPRE measurements. Using ubiquitin as a model system, we measured the sPRE effect from a generation 5 PAMAM dendrimer (G5-Gd) as a function of temperature and pH and compared to conventional relaxation agents. We also demonstrated the utility of G5-Gd in sPRE studies to monitor changes in the structures of two proteins as they bind their ligands. These studies highlight the attractive properties of these macromolecular relaxation agents in biomolecular sPRE.

The use of Escherichia coli for recombinant protein production is a cornerstone in structural biology, particularly for nuclear magnetic resonance (NMR) spectroscopy studies. Understanding the metabolic behavior of E. coli under different carbon sources is critical for optimizing isotope labeling strategies, which are essential for protein structure determination by NMR. Recent advancements, such as mixed pyruvate labeling, have enabled improved backbone resonance assignment in large proteins, making selective isotopic labeling strategies more important than ever for NMR studies. In this study, we aimed to investigate the metabolic adaptations of E. coli when grown on pyruvate as the sole carbon source, a common condition used to achieve selective labeling for NMR spectroscopy. Using NMR-based metabolomics, we tracked key metabolic shifts throughout the culture process to better understand how pyruvate metabolism affects protein production and isotopic labeling. Our results reveal that pyruvate is rapidly depleted before IPTG induction, while acetate and lactate accumulate due to overflow metabolism. These byproducts persist after induction, indicating that pyruvate is diverted into waste pathways, which limits its efficient use in isotope incorporation. This metabolic inefficiency presents a challenge for isotopic labeling protocols that rely on pyruvate as a carbon source for NMR studies. Our results highlight the need to fine-tune pyruvate supplementation to improve metabolic efficiency and isotopic labeling, making this study directly relevant to optimizing protocols for NMR studies involving protein structure determination. These insights provide valuable guidance for enhancing the quality and yield of isotopically labeled proteins in NMR spectroscopy.

Addition of glycine betaine up to 1 M gave rise to increased intensity for some weak signals in the HSQC spectra of barnase and Plasmodium falciparum flap endonuclease. The signals that were enhanced were low intensity signals, often from amide groups with rapid internal motion (low order parameter). The majority of signals are however somewhat weaker because of the increased viscosity. Addition of betaine is shown to cause a small but significant overall increase in order parameter, no consistent effect on conformational change on the µs-ms timescale, and a reduction in amide exchange rates, by a factor of about 3. The results are consistent with a model in which betaine leads to a reduction in fluctuations within the bulk water, which in turn produces a reduction in internal fluctuations of the protein.

Structurally diverse ensembles of intrinsically disordered proteins or regions are difficult to determine, because experimental observables usually report a conformational average. Therefore, in order to infer the underlying distribution, a set of experiments that measure different aspects of the system is necessary. In principle, there exists a set of cross-correlated relaxation (CCR) rates that report on protein backbone geometry in a complementary way. However, CCR rates are hard to interpret, because geometric information is encoded in an ambiguous way and they present themselves as a convolute of both structure and dynamics. Despite these challenges, CCR rates analyzed within a suitable statistical framework are able to identify conformations in structured proteins. In the context of disordered proteins, we find that this approach has to be adjusted to account for local dynamics via including an additional CCR rate. The results of this study show that CCR rates can be used to characterize structure propensities also in disordered proteins. Instead of using an experimental reference structure, we employed computational spectroscopy to calculate CCR rates from molecular dynamics (MD) simulations and subsequently compared the results to conformations as observed directly in the MD trajectory.

NMR is a powerful tool for the structural and dynamic study of proteins. One of the necessary conditions for the study of these proteins is their isotopic labelling with N and C. One of the most widely used methods to obtain these labelled proteins is heterologous expression of the proteins in E. coli using C-D-glucose and NHCl as the sole nutrient sources. In recent years, the price of C-D-glucose has almost tripled, making it essential to develop labelling methods that are as cost effective as possible. In this work, different parameters were studied to achieve the most rational use of C-D-glucose, and an optimized method was developed to obtain labelled proteins with high labelling and low C-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth.

Paramagnetic probes provide long-range distance information and report on minor conformations of biomacromolecules. However, it is important to realize that any probe can affect the system of interest. Here, we report on the effects of attaching a small nitroxide spin label [TEMPO, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl] to xylotriose, a substrate of the enzyme xylanase from Bacillus circulans (BcX). BcX has a long and narrow active site cleft accommodating six xylose units and a secondary binding site on its surface. The aim of the study was to probe the interactions of the substrate with the enzyme using paramagnetic relaxation enhancements (PREs). Binding of the substrate to the surface exposed secondary binding site resulted in strong and localized PREs, indicative of well-defined binding. The xylotriose with diamagnetic control tag was still able to bind the active site cleft, though the rate of exchange was reduced relative to that of untagged xylotriose. The substrate with the paramagnetic TEMPO was not able to bind inside the active site cleft. Also, additional interactions on another surface location showed differences between the paramagnetic substrate and the diamagnetic control, despite the minimal chemical differences between TEMPO modified xylotriose and its reduced, diamagnetic counterpart. Our findings underscore the sensitivity of BcX substrate binding to minor substrate modifications. This study serves as a reminder that any probe, including the attachment of a small paramagnetic group, can affect the behavior of the system under investigation. Even the chemical difference between a paramagnetic tag and its diamagnetic control can result in differences in the molecular interactions.

The investigation of structural propensities of proteins is essential for understanding how they function at the molecular level. NMR, offering atomic-scale information, is often the method of choice. One of the available techniques relies on the cross-correlated relaxation (CCR) effect, whose magnitude is related to local spatial conformation. Application of these methods is difficult if the protein under investigation exhibits high mobility, because NMR observables like CCR rates and chemical shifts present themselves as mere averages of an underlying ensemble distribution. Furthermore, relaxation observables are a convolution of structural and dynamical components. Despite these challenges, it is possible to infer the underlying structural ensemble by combining information from several CCR rates with a different geometrical dependence. In this paper, we present a set of eight CCR experiments tailored for proteins of a highly dynamic nature. Analyzed together, they yield a distribution of backbone dihedral angles for each residue of the protein. The experiments were validated on the folded protein ubiquitin using PDB-deposited NMR structures for comparison. Extraordinary peak separation, achieved by evolving four different chemical shifts, allows for the application of this method to intrinsically disordered proteins in future studies.