Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts of their metabolism. The main drawbacks of commonly used matrices include the inability to recover intact cells post-measurement for additional analyses and/or requirements for specific operating temperatures. Here, we report on the development and characterization of a set of thermosensitive and nontoxic triblock copolymers based on poly(D,L-lactide)-b-poly(ethylene glycol)-b-poly(D,L-lactide) (PLA-PEG-PLA). Here, we show for the first time that these copolymers are suitable as immobilizer matrices for the acquisition of in-cell NMR spectra of nucleic acids and proteins over a commonly used sample temperature range of 15-40 °C and, importantly, allow recovery of cells after completion of in-cell NMR spectra acquisition. We compared the performances of currently used matrices in terms of cell viability (dye exclusion assays), cellular metabolism (1D 31P NMR), and quality of in-cell NMR spectra of two model biomacromolecules (hybrid double-stranded/i-motif DNA and ubiquitin). Our results demonstrate the suitability and advantages of PLA-PEG-PLA copolymers for application in bioreactor-assisted in-cell NMR.

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-β arrangement rich in β-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those of flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (≥ 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of ε ~ 50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε ~ 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.

Fragment-based drug discovery (FBDD) and validation of small molecule binders using NMR spectroscopy is an established and widely used method in the early stages of drug discovery. Starting from a library of small compounds, ligand- or protein-observed NMR methods are employed to detect binders, typically weak, that become the starting points for structure-activity relationships (SAR) by NMR. Unlike the more frequently used ligand-observed 1D NMR techniques, protein-observed 2D 1H-15N or 1H-13C heteronuclear correlation (HSQC or HMQC) methods offer insights that include the mechanism of ligand engagement on the target and direct binding affinity measurements in addition to routine screening. We hereby present the development of a set of software tools within the MestReNova (Mnova) package for analyzing 2D NMR for FBDD and hit validation purposes. The package covers three main tasks: (1) unsupervised profiling of raw data to identify outlier data points to exclude in subsequent analyses; (2) batch processing of single-point spectra to identify and rank binders based on chemical shift perturbations or spectral peak intensity changes; and (3) batch processing of multiple titration series to derive binding affinities (KD) by tracing the changes in peak locations or measuring global spectral changes. Toward this end, we implemented and evaluated a set of algorithms for automated peak tracing, spectral binning, and variance analysis by PCA, and a new tool for spectral data intensity comparison using ECHOS. The accuracy and speed of the tools are demonstrated on 2D NMR binding data collected on ligands used in the development of potential inhibitors of the anti-apoptotic MCL-1 protein.

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered.

Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative "Stablelabel" containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in "Stablelabel" lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.

In the last three decades, the scope of solid-state NMR has expanded to exploring complex biomolecules, from large protein assemblies to intact cells at atomic-level resolution. This diversity in macromolecules frequently features highly flexible components whose insoluble environment precludes the use of solution NMR to study their structure and interactions. While High-resolution Magic-Angle Spinning (HR-MAS) probes offer the capacity for gradient-based 1H-detected spectroscopy in solids, such probes are not commonly used for routine MAS NMR experiments. As a result, most exploration of the flexible regime entails either 13C-detected experiments, the use of partially perdeuterated systems, or ultra-fast MAS. Here we explore proton-detected pulse schemes probing through-bond 13C-13C networks to study mobile protein sidechains as well as polysaccharides in a broadband manner. We demonstrate the use of such schemes to study a mixture of microtubule-associated protein (MAP) tau and human microtubules (MTs), and the cell wall of the fungus Schizophyllum commune using 2D and 3D spectroscopy, to show its viability for obtaining unambiguous correlations using standard fast-spinning MAS probes at high and ultra-high magnetic fields.

NMR spectroscopy is an excellent tool for studying protein structure and dynamics which provides a deeper understanding of biological function. As the size of the biomolecule of interest increases, it can become advantageous to dilute the number of observed signals in the NMR spectrum to decrease spectral overlap and increase resolution. One way to limit the number of resonances in the NMR data is by selectively labeling a smaller domain within the larger macromolecule, a process called segmental isotopic labeling. Many examples of segmental isotopic labeling have been described where two segments of a protein are ligated together by chemical or enzymatic means, but there are far fewer descriptions of a three or more segment ligation reaction. Herein, we describe an enzymatic segmental labeling scheme that combines the widely used Sortase A and more recently described OaAEP1 for a two site ligation strategy. In preparation to study proposed long-range allostery in the 104 kDa DNA damage repair protein Rad50, we ligated side-chain methyl group labeled Zn Hook domain between two long segments of otherwise unlabeled P.furiosus Rad50. Enzymatic activity data demonstrated that the scars resulting from the ligation reactions did not affect Rad50 function within the Mre11-Rad50 DNA double strand break repair complex. Finally, methyl-based NMR spectroscopy confirmed the formation of the full-length ligated protein. Our strategy highlights the strengths of OaAEP1 for segmental labeling, namely faster reaction times and a smaller recognition sequence, and provides a straightforward template for using these two enzymes in multisite segmental labeling reactions.

The nuclear Overhauser effect (NOE) is one of NMR spectroscopy's most important and versatile parameters. NOE is routinely utilized to determine the structures of medium-to-large size biomolecules and characterize protein-protein, protein-RNA, protein-DNA, and protein-ligand interactions in aqueous solutions. Typical [1H,1H] NOESY pulse sequences incorporate water suppression schemes to reduce the water signal that dominates 1H-detected spectra and minimize NOE intensity losses due to unwanted polarization exchange between water and labile protons. However, at high- and ultra-high magnetic fields, the excitation of the water signal during the execution of the NOESY pulse sequences may cause significant attenuation of NOE cross-peak intensities. Using an evolutionary algorithm coupled with artificial intelligence, we recently designed high-fidelity pulses [Water irrAdiation DEvoid (WADE) pulses] that elude water excitation and irradiate broader bandwidths relative to commonly used pulses. Here, we demonstrate that WADE pulses, implemented into the 2D [1H,1H] NOESY experiments, increase the intensity of the NOE cross-peaks for labile and, to a lesser extent, non-exchangeable protons. We applied the new 2D [1H,1H] WADE-NOESY pulse sequence to two well-folded, medium-size proteins, i.e., the K48C mutant of ubiquitin and the Raf kinase inhibitor protein. We observed a net increase of the NOE intensities varying from 30 to 170% compared to the commonly used NOESY experiments. The new WADE pulses can be easily engineered into 2D and 3D homo- and hetero-nuclear NOESY pulse sequences to boost their sensitivity.

The accelerated acquisition of multidimensional NMR spectra using sparse non-uniform sampling (NUS) has been widely adopted in recent years. The key concept in NUS is that a major part of the data is omitted during measurement, and then reconstructed using, for example, compressed sensing (CS) methods. CS requires spectra to be compressible, that is, they should contain relatively few "significant" points. The more compressible the spectrum, the fewer experimental NUS points needed in order for it to be accurately reconstructed. In this paper we show that the CS processing of similar spectra can be enhanced by reconstructing only the differences between them. Accurate reconstruction can be obtained at lower sampling levels as the difference is sparser than the spectrum itself. In many situations this method is superior to "conventional" compressed sensing. We exemplify the concept of "difference CS" with one such case-the study of alpha-synuclein binding to liposomes and its dependence on temperature. To obtain information on temperature-dependent transitions between different states, we need to acquire several dozen spectra at various temperatures, with and without the presence of liposomes. Our detailed investigation reveals that changes in the binding modes of the alpha-synuclein ensemble are not only temperature-dependent but also show non-linear behavior in their transitions. Our proposed CS processing approach dramatically reduces the number of NUS points required and thus significantly shortens the experimental time.

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of 13C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [13C,1H]-HSQC cross-peaks free of one-bond 13C-13C couplings. Selective 13C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.

In protein nuclear magnetic resonance (NMR), chemical shift assignment provides a wealth of information. However, acquisition of high-quality solid-state NMR spectra depends on protein-specific dynamics. For membrane proteins, bilayer heterogeneity further complicates this observation. Since the efficiency of cross-polarization transfer is strongly entwined with protein dynamics, optimal temperatures for spectral sensitivity and resolution will depend not only on inherent protein dynamics, but temperature-dependent phase properties of the bilayer environment. We acquired 1-, 2-, and 3D homo- and heteronuclear experiments of the chemokine receptor CCR3 in a 7:3 phosphatidylcholine:cholesterol lipid environment. 1D direct polarization, cross polarization (CP), and T2' experiments indicate sample temperatures below - 25 °C facilitate higher CP enhancement and longer-lived transverse relaxation times. T1rho experiments indicate intermediate timescales are minimized below a sample temperature of - 20 °C. 2D DCP NCA experiments indicated optimal CP efficiency and resolution at a sample temperature of - 30 °C, corroborated by linewidth analysis in 3D NCACX at - 30 °C compared to - 5 °C. This optimal temperature is concluded to be directly related the lipid phase transition, measured to be between - 20 and 15 °C based on rINEPT signal of all-trans and trans-gauche lipid acyl conformations. Our results have critical implications in acquisition of SSNMR membrane protein assignment spectra, as we hypothesize that different lipid compositions with different phase transition properties influence protein dynamics and therefore the optimal acquisition temperature.

Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol-water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol-water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains-transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP.

A methyl Transverse Relaxation Optimized Spectroscopy (methyl-TROSY) based, multiple quantum (MQ) 13C Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiment is described. The experiment is derived from the previously developed MQ 13C-1H CPMG scheme (Korzhnev in J Am Chem Soc 126: 3964-73, 2004) supplemented with a CPMG train of refocusing 1H pulses applied with constant frequency and synchronized with the 13C CPMG pulse train. The optimal 1H 'decoupling' scheme that minimizes the amount of fast-relaxing methyl MQ magnetization present during CPMG intervals, makes use of an XY-4 phase cycling of the refocusing composite 1H pulses. For small-to-medium sized proteins, the MQ 13C CPMG experiment has the advantage over its single quantum (SQ) 13C counterpart of significantly reducing intrinsic, exchange-free relaxation rates of methyl coherences. For high molecular weight proteins, the MQ 13C CPMG experiment eliminates complications in the interpretation of MQ 13C-1H CPMG relaxation dispersion profiles arising from contributions to exchange from differences in methyl 1H chemical shifts between ground and excited states. The MQ 13C CPMG experiment is tested on two protein systems: (1) a triple mutant of the Fyn SH3 domain that interconverts slowly on the chemical shift time scale between the major folded state and an excited state folding intermediate; and (2) the 82-kDa enzyme Malate Synthase G (MSG), where chemical exchange at individual Ile δ1 methyl positions occurs on a much faster time-scale.

NMR isotope shifts occur due to small differences in nuclear shielding when nearby atoms are different isotopes. For molecules dissolved in 1:1 H2O:D2O, the resulting mixture of N-H and N-D isotopes leads to a small splitting of resonances from adjacent nuclei. We used multidimensional NMR to measure isotope shifts for the proteins CUS-3iD and CspA. We observed four-bond 4∆N(ND) isotope shifts in high-resolution 2D 15N-TROSY experiments of the perdeuterated proteins that correlate with the torsional angle psi. Three-bond 3∆C'(ND) isotope shifts detected in H(N)CO spectra correlate with the intraresidue H-O distance, and to a lesser extent with the dihedral angle phi. The conformational dependence of the isotope shifts agree with those previously reported in the literature. Both the 4∆N(ND) and 3∆C'(ND) isotope shifts are sensitive to distances between the atoms giving rise to the isotope shifts and the atoms experiencing the splitting, however, these distances are strongly correlated with backbone dihedral angles making it difficult to resolve distance from stereochemical contributions to the isotope shift. H(NCA)CO spectra were used to measure two-bond 2∆C'(ND) isotope shifts and [D]/[H] fractionation factors. Neither parameter showed significant differences for hydrogen-bonded sites, or changes over a 25° temperature range, suggesting they are not sensitive to hydrogen bonding. Finally, the quartet that arises from the combination of 2∆C'(ND) and 3∆C'(ND) isotope shifts in H(CA)CO spectra was used to measure synchronized hydrogen exchange for the sequence neighbors A315-S316 in the protein CUS-3iD. In many of our experiments we observed minor resonances due to the 10% D2O used for the sample deuterium lock, indicating isotope shifts can be a source of spectral heterogeneity in standard NMR experiments. We suggest that applications of isotope shifts such as conformational analysis and correlated hydrogen exchange could benefit from the larger magnetic fields becoming available.

A single experimental method alone often fails to provide the resolution, accuracy, and coverage needed to model integral membrane proteins (IMPs). Integrating computation with experimental data is a powerful approach to supplement missing structural information with atomic detail. We combine RosettaNMR with experimentally-derived paramagnetic NMR restraints to guide membrane protein structure prediction. We demonstrate this approach using the disulfide bond formation protein B (DsbB), an α-helical IMP. Here, we attached a cyclen-based paramagnetic lanthanide tag to an engineered non-canonical amino acid (ncAA) using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction. Using this tagging strategy, we collected 203 backbone HN pseudocontact shifts (PCSs) for three different labeling sites and used these as input to guide de novo membrane protein structure prediction protocols in Rosetta. We find that this sparse PCS dataset combined with 44 long-range NOEs as restraints in our calculations improves structure prediction of DsbB by enhancements in model accuracy, sampling, and scoring. The inclusion of this PCS dataset improved the Cα-RMSD transmembrane segment values of the best-scoring and best-RMSD models from 9.57 Å and 3.06 Å (no NMR data) to 5.73 Å and 2.18 Å, respectively.

Over the last decade amide 15N CEST experiments have emerged as a popular tool to study protein dynamics that involves exchange between a 'visible' major state and sparsely populated 'invisible' minor states. Although initially introduced to study exchange between states that are in slow exchange with each other (typical exchange rates of, 10 to 400 s-1), they are now used to study interconversion between states on the intermediate to fast exchange timescale while still using low to moderate (5 to 350 Hz) 'saturating' B1 fields. The 15N CEST experiment is very sensitive to exchange as the exchange delay TEX can be quite long (~0.5 s) allowing for a large number of exchange events to occur making it a very powerful tool to detect minor sates populated ([Formula: see text]) to as low as 1%. When systems are in fast exchange and the 15N CEST data has to be described using a model that contains exchange, the exchange parameters are often poorly defined because the [Formula: see text] versus [Formula: see text] and [Formula: see text] versus exchange rate ([Formula: see text]) plots can be quite flat with shallow or no minima and the analysis of such 15N CEST data can lead to wrong estimates of the exchange parameters due to the presence of 'spurious' minima. Here we show that the inclusion of experimentally derived constraints on the intrinsic transverse relaxation rates and the inclusion of visible state peak-positions during the analysis of amide 15N CEST data acquired with moderate B1 values (~50 to ~350 Hz) results in convincing minima in the [Formula: see text] versus [Formula: see text] and the [Formula: see text] versus [Formula: see text] plots even when exchange occurs on the 100 μs timescale. The utility of this strategy is demonstrated on the fast-folding Bacillus stearothermophilus peripheral subunit binding domain that folds with a rate constant ~104 s-1. Here the analysis of 15N CEST data alone results in [Formula: see text] versus [Formula: see text] and [Formula: see text] versus [Formula: see text] plots that contain shallow minima, but the inclusion of visible-state peak positions and restraints on the intrinsic transverse relaxation rates of both states during the analysis of the 15N CEST data results in pronounced minima in the [Formula: see text] versus [Formula: see text] and [Formula: see text] versus [Formula: see text] plots and precise exchange parameters even in the fast exchange regime ([Formula: see text]~5). Using this strategy we find that the folding rate constant of PSBD is invariant (~10,500 s-1) from 33.2 to 42.9 °C while the unfolding rates (~70 to ~500 s-1) and unfolded state populations (~0.7 to ~4.3%) increase with temperature. The results presented here show that protein dynamics occurring on the 10 to 104 s-1 timescale can be studied using amide 15N CEST experiments.