Journal of Biomolecular NMR最新文献

The dynamics of the backbone and side-chains of protein are routinely studied by interpreting experimentally determined ¹⁵N spin relaxation rates. R₁(¹⁵N), the longitudinal relaxation rate, reports on fast motions and encodes, together with the transverse relaxation R₂, structural information about the shape of the molecule and the orientation of the amide bond vectors in the internal diffusion frame. Determining error-free ¹⁵N longitudinal relaxation rates remains a challenge for small, disordered, and medium-sized proteins. Here, we show that mono-exponential fitting is sufficient, with no statistical preference for bi-exponential fitting up to 800 MHz. A detailed comparison of the TROSY and HSQC techniques at medium and high fields showed no statistically significant differences. The least error-prone DD/CSA interference removal technique is the selective inversion of amide signals while avoiding water resonance. The exchange of amide with solvent deuterons appears to affect the rate R₁ of solvent-exposed amides in all fields tested and in each DD/CSA interference removal technique in a statistically significant manner. In summary, the most accurate R₁(¹⁵N) rates in proteins are achieved by selective amide inversion, without the addition of D₂O. Importantly, at high magnetic fields stronger than 800 MHz, when non-mono-exponential decay is involved, it is advisable to consider elimination of the shortest delays (typically up to 0.32 s) or bi-exponential fitting.

Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. ¹H,¹³C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.

Understanding the structure and function of nucleic acids in their native environment is crucial to structural biology and one focus of in-cell NMR spectroscopy. Many challenges hamper in-cell NMR in human cell lines, e.g. sample decay through cell death and RNA degradation. The resulting low signal intensities and broad line widths limit the use of more complex NMR experiments, reducing the possible structural and dynamic information that can be extracted. Here, we optimize the detection of imino proton signals, indicators of base-pairing and therefore secondary structure, of a double-stranded DNA oligonucleotide in HeLa cells, using selective excitation. We demonstrate the reproducible quantification of in-cell selective longitudinal relaxation times (selT₁), which are reduced compared to the in vitro environment, as a result of interactions with the complex cellular environment. By measuring the intracellular selT_1, we optimize the existing proton pulse sequences, and shorten measurement time whilst enhancing the signal gained per unit of time. This exemplifies an advantage of selective excitation over conventional methods like jump-return water suppression for in-cell NMR. Furthermore, important experimental controls are discussed, including intracellular quantification, supernatant control measurements, as well as the processing of lowly concentrated in-cell NMR samples. We expect that robust and fast in-cell NMR experiments of nucleic acids will facilitate the study of structure and dynamics and reveal their functional correlation.

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH₂ groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing ¹⁵N and ¹H components of NH₂ multiplets. Although even modest sensitivity gains in 2D NH₂-TROSY correlation maps compared to their decoupled NH₂-HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH₂ sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH₂-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH₂ positions of the L99A T4 lysozyme mutant - a model system for studies of the role of protein dynamics in ligand binding.

Fluorine (¹⁹F) NMR is emerging as an invaluable analytical technique in chemistry, biochemistry, structural biology, material science, drug discovery, and medicine, especially due to the inherent rarity of naturally occurring fluorine in biological, organic, and inorganic compounds. Here, we revisit the under-reported problem of fluoride leaching from new and unused glass NMR tubes. We characterised the leaching of free fluoride from various types of new and unused glass NMR tubes over the course of several hours and quantify this contaminant to be at micromolar concentrations for typical NMR sample volumes across multiple glass types and brands. We find that this artefact is undetectable for samples prepared in quartz NMR tubes within the timeframes of our experiments. We also observed that pre-soaking new glass NMR tubes combined with rinsing removes this contamination below micromolar levels. Given the increasing popularity of ¹⁹F NMR across a wide range of fields, increasing popularity of single-use screening tubes, the long collection times required for relaxation studies and samples of low concentrations, and the importance of avoiding contamination in all NMR experiments, we anticipate that our simple solution will be useful to biomolecular NMR spectroscopists.

Solution NMR spectroscopy is a particularly powerful technique for characterizing the functional dynamics of biomolecules, which is typically achieved through the quantitative characterization of chemical exchange processes via the measurement of spin relaxation rates. In addition to the conventional nuclei such as ¹⁵N and ¹³C, which are abundant in biomolecules, fluorine-19 (¹⁹F) has recently garnered attention and is being widely used as a site-specific spin probe. While ¹⁹F offers the advantages of high sensitivity and low background, it can be susceptible to artifacts in quantitative relaxation analyses due to a multitude of dipolar and scalar coupling interactions with nearby ¹H spins. In this study, we focused on the ribose 2'-¹⁹F spin probe in nucleic acids and investigated the effects of ¹H-¹⁹F spin interactions on the quantitative characterization of slow exchange processes on the millisecond time scale. We demonstrated that the ¹H-¹⁹F dipolar coupling can significantly affect the interpretation of ¹⁹F chemical exchange saturation transfer (CEST) experiments when ¹H decoupling is applied, while the ¹H-¹⁹F interactions have a lesser impact on Carr-Purcell-Meiboom-Gill relaxation dispersion applications. We also proposed a modified CEST scheme to alleviate these artifacts along with experimental verifications on self-complementary RNA systems. The theoretical framework presented in this study can be widely applied to various ¹⁹F spin systems where ¹H-¹⁹F interactions are operative, further expanding the utility of ¹⁹F relaxation-based NMR experiments.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Deuterium (²H) spin relaxation of ¹³CH₂D methyl groups has been widely applied to investigate picosecond-to-nanosecond conformational dynamics in proteins by solution-state NMR spectroscopy. The B₀ dependence of the ²H spin relaxation rates is represented by a linear relationship between the spectral density function at three discrete frequencies J(0), J(ω_D) and J(2ω_D). In this study, the linear relation between ²H relaxation rates at B₀ fields separated by a factor of two and the interpolation of rates at intermediate frequencies are combined for a more robust approach for spectral density mapping. The general usefulness of the approach is demonstrated on a fractionally deuterated (55%) and alternate ¹³C-¹²C labeled sample of E. coli RNase H. Deuterium relaxation rate constants (R₁, R_1ρ, R_Q, R_AP) were measured for 57 well-resolved ¹³CH₂D moieties in RNase H at ¹H frequencies of 475 MHz, 500 MHz, 900 MHz, and 950 MHz. The spectral density mapping of the 475/950 MHz data combination was performed independently and jointly to validate the expected relationship between data recorded at B₀ fields separated by a factor of two. The final analysis was performed by jointly analyzing 475/950 MHz rates with 700 MHz rates interpolated from 500/900 MHz data to yield six J(ω_D) values for each methyl peak. The J(ω) profile for each peak was fit to the original (τ_M, S_f², τ_f) or extended model-free function (τ_M, S_f², S_s², τ_f, τ_s) to obtain optimized dynamic parameters.

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

The fast motions of proteins at the picosecond to nanosecond timescale, known as fast dynamics, are closely related to protein conformational entropy and rearrangement, which in turn affect catalysis, ligand binding and protein allosteric effects. The most used NMR approach to study fast protein dynamics is the model free method, which uses order parameter S² to describe the amplitude of the internal motion of local group. However, to obtain order parameter through NMR experiments is quite complex and lengthy. In this paper, we present a machine learning approach for predicting backbone ¹H-¹⁵N order parameters based on protein NMR structure ensemble. A random forest model is used to learn the relationship between order parameters and structural features. Our method achieves high accuracy in predicting backbone ¹H-¹⁵N order parameters for a test dataset of 10 proteins, with a Pearson correlation coefficient of 0.817 and a root-mean-square error of 0.131.