Journal of Biomolecular NMR最新文献

{"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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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}

{"title":"Decorating phenylalanine side-chains with triple labeled 13C/19F/2H isotope patterns","authors":"Giorgia Toscano,&nbsp;Julian Holzinger,&nbsp;Benjamin Nagl,&nbsp;Georg Kontaxis,&nbsp;Hanspeter Kählig,&nbsp;Robert Konrat,&nbsp;Roman J. Lichtenecker","doi":"10.1007/s10858-024-00440-z","DOIUrl":"10.1007/s10858-024-00440-z","url":null,"abstract":"<div><p>We present an economic and straightforward method to introduce ¹³C-¹⁹F spin systems into the deuterated aromatic side chains of phenylalanine as reporters for various protein NMR applications. The method is based on the synthesis of [4-¹³C, 2,3,5,6-²H₄] 4-fluorophenylalanine from the commercially available isotope sources [2-¹³C] acetone and deuterium oxide. This compound is readily metabolized by standard <i>Escherichia coli</i> overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-024-00440-z.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140178941","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":"Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR","authors":"Bharat P. Chaudhary,&nbsp;Jochem Struppe,&nbsp;Hem Moktan,&nbsp;David Zoetewey,&nbsp;Donghua H. Zhou,&nbsp;Smita Mohanty","doi":"10.1007/s10858-024-00437-8","DOIUrl":"10.1007/s10858-024-00437-8","url":null,"abstract":"<div><p><i>N</i>-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in <i>N</i>-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein. Oligosaccharyltransferase (OST), a multi-subunit membrane embedded enzyme catalyzes this glycosylation reaction in eukaryotes. In yeast, Ost4 is the smallest of nine subunits and bridges the interaction of the catalytic subunit, Stt3, with Ost3 (or its homolog, Ost6). Mutations of any C-terminal hydrophobic residues in Ost4 to a charged residue destabilizes the enzyme and negatively impacts its function. Specifically, the V23D mutation results in a temperature-sensitive phenotype in yeast. Here, we report the reconstitution of both purified recombinant Ost4 and Ost4V23D each in a POPC/POPE lipid bilayer and their resonance assignments using heteronuclear 2D and 3D solid-state NMR with magic-angle spinning. The chemical shifts of Ost4 changed significantly upon the V23D mutation, suggesting a dramatic change in its chemical environment.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-02-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139988971","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":"Fluorine labelling for in situ 19F NMR in oriented systems","authors":"Kieran T. Cockburn,&nbsp;Brian D. Sykes","doi":"10.1007/s10858-024-00438-7","DOIUrl":"10.1007/s10858-024-00438-7","url":null,"abstract":"<div><p>The focus of this project is to take advantage of the large NMR chemical shift anisotropy of ¹⁹F to determine the orientation of fluorine labeled biomolecules in situ in oriented biological systems such as muscle. The difficulty with a single fluorine atom is that the orientation determined from a chemical shift is not singlevalued in the case of a fully anisotropic chemical shift tensor. The utility of a labeling approach with two fluorine labels in a fixed molecular framework where one of the labels has an axially symmetric chemical shift anisotropy such as a CF₃ group and the other has a fully asymmetric chemical shift anisotropy such as 5-fluorotryptophan is evaluated. The result is that the orientation of the label can be determined straightforwardly from a single one-dimensional ¹⁹F NMR spectrum. The potential applications are widespread and not limited to biological applications.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139968236","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":"A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy","authors":"Ganesh P. Subedi,&nbsp;Elijah T. Roberts,&nbsp;Alexander R. Davis,&nbsp;Paul G. Kremer,&nbsp;I. Jonathan Amster,&nbsp;Adam W. Barb","doi":"10.1007/s10858-023-00434-3","DOIUrl":"10.1007/s10858-023-00434-3","url":null,"abstract":"<div><p>A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[¹⁵N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for ¹⁵N-labeling in HEK293 cells. </p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139968748","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":"Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale","authors":"Ved Prakash Tiwari,&nbsp;Debajyoti De,&nbsp;Nemika Thapliyal,&nbsp;Lewis E. Kay,&nbsp;Pramodh Vallurupalli","doi":"10.1007/s10858-023-00431-6","DOIUrl":"10.1007/s10858-023-00431-6","url":null,"abstract":"<div><p>Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a ‘visible’ major conformer exchanges with two ‘invisible’ minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate—fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide ¹⁵N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although ¹⁵N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, ¹⁵N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of ¹⁵N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale (<span>({k}_{ex,FI1})</span> ~ 550 s⁻¹, <span>({k}_{ex,FI2})</span> ~ 1200 s⁻¹, <span>({k}_{ex,I1I2})</span> ~ 5000 s⁻¹), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15 °C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139085430","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}