Journal of Biomolecular NMR最新文献

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.

A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D₂O and ²H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D₂O) or deuterated glucose is not required to obtain ¹H–¹³C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100–150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.

In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, ¹⁹F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The ¹⁹F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the “toggle switch tryptophan”. Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.

Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [¹⁵N, ¹³C, ²H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in ¹H₂O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of ¹H_N, ¹⁵N, ¹³C′, ¹³C_α, ¹³C_β resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs’ higher order structures, dynamics and interactions, using solution-state NMR.