Journal of Biomolecular NMR最新文献

{"title":"Transverse relaxation optimized spectroscopy of NH2 groups in glutamine and asparagine side chains of proteins","authors":"Vitali Tugarinov,&nbsp;Francesco Torricella,&nbsp;Jinfa Ying,&nbsp;G. Marius Clore","doi":"10.1007/s10858-024-00445-8","DOIUrl":"10.1007/s10858-024-00445-8","url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 4","pages":"199 - 213"},"PeriodicalIF":1.3,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-024-00445-8.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141854475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Micromolar fluoride contamination arising from glass NMR tubes and a simple solution for biomolecular applications","authors":"Khushboo Matwani,&nbsp;Jasmine Cornish,&nbsp;Erika Alden DeBenedictis,&nbsp;Gabriella T. Heller","doi":"10.1007/s10858-024-00442-x","DOIUrl":"10.1007/s10858-024-00442-x","url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 3","pages":"161 - 167"},"PeriodicalIF":1.3,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-024-00442-x.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141776891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative analysis of the slow exchange process by 19F NMR in the presence of scalar and dipolar couplings: applications to the ribose 2′-19F probe in nucleic acids","authors":"Yuki Toyama,&nbsp;Ichio Shimada","doi":"10.1007/s10858-024-00446-7","DOIUrl":"10.1007/s10858-024-00446-7","url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 4","pages":"215 - 235"},"PeriodicalIF":1.3,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141449249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspects","authors":"Dallas Bell,&nbsp;Florian Lindemann,&nbsp;Lisa Gerland,&nbsp;Hanna Aucharova,&nbsp;Alexander Klein,&nbsp;Daniel Friedrich,&nbsp;Matthias Hiller,&nbsp;Kristof Grohe,&nbsp;Tobias Meier,&nbsp;Barth van Rossum,&nbsp;Anne Diehl,&nbsp;Jon Hughes,&nbsp;Leonard J. Mueller,&nbsp;Rasmus Linser,&nbsp;Anne-Frances Miller,&nbsp;Hartmut Oschkinat","doi":"10.1007/s10858-024-00444-9","DOIUrl":"10.1007/s10858-024-00444-9","url":null,"abstract":"<div><p>Solution NMR is typically applied to biological systems with molecular weights &lt; 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 <i>Sulfolobus acidocaldarius</i> bifurcating electron transfer flavoprotein (<i>Sa</i>ETF), tryptophan synthases from <i>Salmonella typhimurium</i> (<i>St</i>TS) and their dimeric β-subunits from <i>Pyrococcus furiosus</i> (<i>Pf</i>TrpB), 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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 3","pages":"179 - 192"},"PeriodicalIF":1.3,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7616530/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141431015","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deuterium spin relaxation of fractionally deuterated ribonuclease H using paired 475 and 950 MHz NMR spectrometers","authors":"Shibani Bhattacharya,&nbsp;Kristen M. Varney,&nbsp;Tassadite Dahmane,&nbsp;Bruce A. Johnson,&nbsp;David J. Weber,&nbsp;Arthur G. Palmer III","doi":"10.1007/s10858-024-00443-w","DOIUrl":"10.1007/s10858-024-00443-w","url":null,"abstract":"<div><p>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 <i>B</i>₀ dependence of the ²H spin relaxation rates is represented by a linear relationship between the spectral density function at three discrete frequencies <i>J</i>(0), <i>J</i>(<i>ω</i>_D) and <i>J</i>(2<i>ω</i>_D). In this study, the linear relation between ²H relaxation rates at <i>B</i>₀ 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 <i>E. coli</i> RNase H. Deuterium relaxation rate constants (<i>R</i>₁, <i>R</i>_1<i>ρ</i>, <i>R</i>_<i>Q</i>, <i>R</i>_<i>AP</i>) 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 <i>B</i>₀ 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 <i>J</i>(<i>ω</i>_D) values for each methyl peak. The <i>J</i>(<i>ω</i>) profile for each peak was fit to the original (<i>τ</i>_<i>M</i>, <i>S</i>_<i>f</i>², <i>τ</i>_<i>f</i>) or extended model-free function (<i>τ</i>_<i>M</i>, <i>S</i>_<i>f</i>², <i>S</i>_<i>s</i>², <i>τ</i>_<i>f</i>, <i>τ</i>_<i>s</i>) to obtain optimized dynamic parameters.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 3","pages":"169 - 177"},"PeriodicalIF":1.3,"publicationDate":"2024-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141295374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Labeling of methyl groups: a streamlined protocol and guidance for the selection of 2H precursors based on molecular weight","authors":"Alexandra Locke,&nbsp;Kylee Guarino,&nbsp;Gordon S. Rule","doi":"10.1007/s10858-024-00441-y","DOIUrl":"10.1007/s10858-024-00441-y","url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 3","pages":"149 - 159"},"PeriodicalIF":1.3,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-024-00441-y.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141086372","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression","authors":"Benxun Pan,&nbsp;Canyong Guo,&nbsp;Dongsheng Liu,&nbsp;Kurt Wüthrich","doi":"10.1007/s10858-024-00439-6","DOIUrl":"10.1007/s10858-024-00439-6","url":null,"abstract":"<div><p>\u0000 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 <i>Pichia pastoris</i>. 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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 3","pages":"133 - 138"},"PeriodicalIF":1.3,"publicationDate":"2024-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140329463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enabling site-specific NMR investigations of therapeutic Fab using a cell-free based isotopic labeling approach: application to anti-LAMP1 Fab","authors":"Arthur Giraud,&nbsp;Lionel Imbert,&nbsp;Adrien Favier,&nbsp;Faustine Henot,&nbsp;Francis Duffieux,&nbsp;Camille Samson,&nbsp;Oriane Frances,&nbsp;Elodie Crublet,&nbsp;Jérôme Boisbouvier","doi":"10.1007/s10858-023-00433-4","DOIUrl":"10.1007/s10858-023-00433-4","url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 2","pages":"73 - 86"},"PeriodicalIF":1.3,"publicationDate":"2024-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140315702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Prediction of order parameters based on protein NMR structure ensemble and machine learning","authors":"Qianqian Wang,&nbsp;Zhiwei Miao,&nbsp;Xiongjie Xiao,&nbsp;Xu Zhang,&nbsp;Daiwen Yang,&nbsp;Bin Jiang,&nbsp;Maili Liu","doi":"10.1007/s10858-024-00435-w","DOIUrl":"10.1007/s10858-024-00435-w","url":null,"abstract":"<div><p>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 <i>S</i>² 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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 2","pages":"87 - 94"},"PeriodicalIF":1.3,"publicationDate":"2024-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140292411","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu","authors":"Whitney N. Costello,&nbsp;Yiling Xiao,&nbsp;Frederic Mentink-Vigier,&nbsp;Jaka Kragelj,&nbsp;Kendra K. Frederick","doi":"10.1007/s10858-024-00436-9","DOIUrl":"10.1007/s10858-024-00436-9","url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 2","pages":"95 - 108"},"PeriodicalIF":1.3,"publicationDate":"2024-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140193061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}