Journal of Biomolecular NMR最新文献

{"title":"Methyl-specific NMR of therapeutic antibodies: cost-effective isotopic labeling strategies in CHO cells for high-resolution structural characterization","authors":"Rida Awad,&nbsp;Arthur Giraud,&nbsp;Béatrice Vibert,&nbsp;Séverine Clavier,&nbsp;Hélène Le Borgne,&nbsp;Laetitia Maçon,&nbsp;Anaïs Muhr-Naninck,&nbsp;Stéphanie Seguin-Huet,&nbsp;Benoit Mothes,&nbsp;Pierre Gans,&nbsp;Oriane Frances,&nbsp;Jérôme Boisbouvier,&nbsp;Elodie Crublet","doi":"10.1007/s10858-026-00492-3","DOIUrl":"10.1007/s10858-026-00492-3","url":null,"abstract":"<div>\u0000 \u0000 <p>Monoclonal antibodies (mAbs) are essential therapeutic agents whose efficacy depends critically on the integrity of their higher-order structure (HOS). Subtle perturbations in HOS, arising from post-translational modifications, chemical degradation or formulation conditions, can impair antigen binding or induce immunogenicity. Nuclear Magnetic Resonance (NMR) spectroscopy, and particularly methyl NMR, offers a powerful means to probe the structure and dynamics of large proteins, such as antibodies, at atomic scale resolution. While 2D ¹H-¹³C methyl correlation spectra at natural abundance provide valuable structural fingerprints, their limited resolution and sensitivity hamper sophisticated NMR studies. Isotopic labeling of methyl groups with ¹³CH₃, combined with tailored deuteration, greatly enhances spectral quality. However, such labeling strategies remain underdeveloped in mammalian systems like CHO cells, which are the standard hosts for therapeutic antibody production. Here, we report an optimized labeling strategy enabling selective incorporation of the ¹³CH₃-labeled methyl groups from the six methyl-bearing amino acids into antibodies expressed in CHO cells. This includes enzymatic synthesis of regio- and stereoselectively labeled isoleucine and valine with partial deuteration and an optimized protocol for their incorporation into CHO-produced proteins. When applied to an anti-LAMP1 therapeutic antibody, this strategy yields highly resolved methyl NMR spectra, enabling comprehensive HOS assessment. It allows the transfer of previously assigned Fab and Fc methyl resonances, resulting in approximately 84% of methyl signals being assigned in the intact mAb. This work establishes a robust framework for NMR-based structural analysis of glycosylated antibodies directly from mammalian expression systems.</p>\u0000 </div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13090208/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147715594","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":"AMIGO - Guided assignment of 13C-methyl labelled proteins","authors":"Thorben Maass,&nbsp;Lorena Rudolph,&nbsp;Thomas Peters,&nbsp;Alvaro Mallagaray","doi":"10.1007/s10858-026-00491-4","DOIUrl":"10.1007/s10858-026-00491-4","url":null,"abstract":"<div>\u0000 \u0000 <p>Over the last 20 years, the number of large proteins accessible to protein-NMR analysis has increased significantly due to the development of selective [¹H_,¹³C]-methyl labelling techniques in combination with methyl-TROSY based NMR experiments. Structure-based strategies for the assignment of [¹H,¹³C]-methyl groups rely on comparing spatial constraints derived from methyl–methyl NOEs to a known three-dimensional structure of the protein of interest. Cross peaks in methyl-TROSY spectra are assigned to specific methyl groups by matching methyl–methyl NOEs, as observed for example in 4D HMQC-NOESY-HMQC spectra, with distances derived from a structural model. This process is commonly referred to as a “methyl walk”. Here, we present AMIGO (Automated Methyl assignment via Iterative Graph Optimization), a novel assignment algorithm that formalises the intuitive methyl walk procedure by constructing graphs with nodes representing specific methyl groups and edges reflecting methyl-methyl NOEs or short methyl-methyl distances in a model. “Building blocks” consisting of nodes and edges are then generated to reconcile structure-based and NOE-based graphs. Assignments are achieved through permutation and concatenation of individual “building blocks” in a modular fashion, enabling efficient computation even for large proteins. Additional experimental restraints, such as paramagnetic relaxation enhancements (PREs) or pseudocontact shifts (PCSs), can be integrated to validate and extend the assignments. The performance of AMIGO was validated using 11 proteins that had previously been assigned and 32 NOE networks that had been generated synthetically.</p>\u0000 </div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13070982/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147669760","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":"Super-Resolution solid-state NMR Spectroscopy","authors":"Olivia Gampp,&nbsp;Riccardo Cadalbert,&nbsp;Roland Riek,&nbsp;Sarah A. Overall","doi":"10.1007/s10858-026-00490-5","DOIUrl":"10.1007/s10858-026-00490-5","url":null,"abstract":"<div><p>Solid-state NMR spectroscopy is often limited by low spectral resolution, a problem typically addressed using fast magic-angle spinning (MAS) and ¹H detection, which require costly specialized hardware. Here, we demonstrate that the super-resolution method—previously applied in solution-state NMR—can be successfully implemented in solid-state NMR to enhance resolution. Applying dynamic number of scans (DNS) sampling to 2D ¹³C-¹³C DARR experiments on the AP205 capsid protein yielded an effective doubling of resolution, halving peak widths from ~ 180 Hz to ~ 87 Hz. Furthermore, DNS acquisition provides a significant advantage over post-acquisition apodization of conventional data with a 20% gain in sensitivity, yielding 309 more detectable peaks with 20% more sequential contacts and 25% more long-range contacts. This method is simple to implement and provides a powerful, accessible strategy to greatly improve the quality of solid-state NMR spectra applicable at all MAS frequencies.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-026-00490-5.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147580124","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":"Optimus peak: automatic peak information extraction from 2D and 3D solution and solid state spectra using evolutionary algorithms","authors":"Rachel E. Brown,&nbsp;Allan R. Willms,&nbsp;Steffen P. Graether","doi":"10.1007/s10858-025-00483-w","DOIUrl":"10.1007/s10858-025-00483-w","url":null,"abstract":"<div>\u0000 \u0000 <p>Peak information extraction is an important step for all types of protein NMR experiments, from structure determination to titration experiments. In most cases, extraction of peak information, or peak-picking, is done semi-automatically or manually, relying on the spectroscopist’s expertise to guard against errors in the algorithm. Optimus Peak is designed for fully automatic peak-picking in multi-dimensional protein NMR spectra for both solid-state and solution NMR. The program shows a low false positive rate, due to its more robust evolutionary algorithm-based fitting. The program is also designed to fit together with other programs by being implemented as a terminal based command line program. Optimus Peak was tested using both solid-state and solution NMR experiments, for both 2D and 3D experiments, using 67 spectra from 12 different proteins, consisting of 26 different types of NMR experiments.</p>\u0000 </div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147430070","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":"Cupriavidus necator, an alternative source for isotopic enrichment of proteins expressed in insect cells for NMR investigations","authors":"Santosh L. Gande,&nbsp;Sridhar Sreeramulu,&nbsp;Krishna Saxena,&nbsp;Fiona Rupprecht,&nbsp;Julian David Langer,&nbsp;Hermann Heumann,&nbsp;Harald Schwalbe","doi":"10.1007/s10858-026-00489-y","DOIUrl":"10.1007/s10858-026-00489-y","url":null,"abstract":"<div><p>Isotopic enrichment of pharmacologically relevant protein targets is crucial for structural studies by nuclear magnetic resonance (NMR) and plays a key role in advancing structure-guided drug discovery. Many clinically important drug targets require expression in eukaryotic systems—such as mammalian, yeast, or insect cells—rather than prokaryotic hosts. This requirement limits the feasibility of high-throughput isotopic labeling and poses challenges for obtaining uniformly isotope-labeled proteins suitable for NMR analysis. While several enrichment strategies have been developed, no broadly applicable enrichment platform has emerged for eukaryotic expression systems. In this study, we introduce <i>Cupriavidus necator</i> as an alternative biological source for ¹⁵N and ¹³C isotopic enrichment to support protein production in eukaryotic systems. To evaluate this approach, we selected the kinase domain of EPHA2, a receptor tyrosine kinase implicated in colorectal cancer progression and an important target for therapeutic inhibitor development. Isotopic incorporation was quantified using liquid chromatography-mass spectrometry (LC-MS), revealing enrichment levels of 79% for ¹⁵N and 69% for ¹³C. These results demonstrate that <i>Cupriavidus necator</i> can serve as a robust and flexible platform for generating isotopically enriched biomolecules compatible with eukaryotic protein expression, thereby enabling NMR investigations of disease-relevant protein targets.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12935839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147281714","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":"Incorporating the BEST methodology in experiments for measuring paramagnetic relaxation enhancements","authors":"Nikolaus M. Loening","doi":"10.1007/s10858-025-00485-8","DOIUrl":"10.1007/s10858-025-00485-8","url":null,"abstract":"<div><p>Band-selective excitation short-transient (BEST) sequences are widely used for protein NMR experiments that start with amide proton magnetization, such as ¹H-¹⁵N HSQC, ¹H-¹⁵N TROSY, and multidimensional backbone assignment experiments, because the optimization of amide proton longitudinal relaxation afforded by the BEST methodology allows for much greater sensitivity when using short scan times. Here we show that the BEST methodology can be easily incorporated in sequences for measuring proton transverse relaxation rates (¹H <i>R</i>₂), which are typically used to determine paramagnetic relaxation enhancements (PREs). The resulting BEST-HSQC-PRE and BEST-TROSY-PRE experiments afford similar or better sensitivity for measuring PREs compared to previous methods, provide equally accurate measurements of transverse relaxation rates (and therefore PREs), and allow shorter scan times to be used.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146211757","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":"Conformation-selective detection of residues in solid proteins under magic-angle-spinning","authors":"Pragyan P. Parida,&nbsp;Pravin P. Taware,&nbsp;Kaustubh R. Mote","doi":"10.1007/s10858-025-00482-x","DOIUrl":"10.1007/s10858-025-00482-x","url":null,"abstract":"<div><p>We demonstrate here a pulse sequence based on rotational-echo double resonance (REDOR) that can help distinguish resonances based on the <span>(psi)</span>-torsion angle, which allows it to distinguish <span>(alpha)</span>-helical and <span>(beta)</span>-sheet regions in solid proteins under magic-angle-spinning. The method relies on conformation-dependent differences in distances between an amide <span>(^{1})</span>H and amide <span>(^{15})</span>N nuclei not covalently attached to it. Dephasing from this remote <span>(^{15})</span>N nucleus is obtained in presence of the much stronger one-bond dipole-dipole coupling by using the <span>(mathrm {theta })</span>-REDOR sequence. Experiments are demonstrated on perdeuterated (and 70% backexchanged) sample of uniformly <span>(^{13})</span>C, <span>(^{15})</span>N labeled model protein GB1 at the magic-angle spinning frequency of 41.67 kHz. This method will be useful in simplifying chemical-shift assignments in proteins where the structure is already known, and we anticipate a direct application in determining secondary structures without relying on <span>(^{13})</span>C chemical-shifts.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12913292/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146211770","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":"Toward site-specific characterization of structural perturbations on glycosylated Fc using NMR at natural abundance","authors":"Béatrice Vibert,&nbsp;Sarah Nguyen,&nbsp;Faustine Henot,&nbsp;Camille Doyen,&nbsp;Oscar Hernandez-Alba,&nbsp;Sarah Cianférani,&nbsp;Séverine Clavier,&nbsp;Oriane Frances,&nbsp;Jérôme Boisbouvier","doi":"10.1007/s10858-025-00484-9","DOIUrl":"10.1007/s10858-025-00484-9","url":null,"abstract":"<div><p>Monoclonal antibodies (mAbs) are leading therapeutic agents due to their high specificity and limited side effects. Ensuring their structural integrity under stress and maintaining batch consistency require robust quality control. Methyl 2D NMR has emerged as a powerful tool to probe mAb structure at natural isotopic abundance, enabling spectral fingerprint comparisons across production batches to detect subtle structural changes. However, extracting atomic-level structural information requires assignment of methyl resonances to their amino acids. While such assignments are available for several antigen-binding fragments (Fabs), no comprehensive assignment has been reported for the crystallisable fragment (Fc). In this study, we present the methyl group assignment of the 50-kDa Fc fragment of an immunoglobulin G1 (IgG1) antibody. Using cell-free expression, strategic isotopic labelling, and high-quality 2D and 3D NMR experiments, we successfully assigned 94% of methyl resonances of a non-glycosylated Fc. Given that therapeutic mAbs are typically produced in Chinese Hamster Ovary (CHO) cells, we transferred this assignment to the methyl spectrum of a glycosylated Fc fragment obtained by the enzymatic cleavage of a CHO-produced mAb at natural abundance, achieving 83% assignment coverage. This assignment was then used to investigate the impact of methionine oxidation on Fc structure at atomic resolution using NMR. The methyl group assignment transforms 2D methyl NMR fingerprinting into a powerful tool for quality control. It enables the direct comparison of spectra acquired on mAbs produced at natural abundance, allowing the detection and localisation of chemical modifications and structural changes without the need for isotopic labelling. This approach offers a robust solution for monitoring the structural integrity of therapeutic antibodies throughout development and manufacturing.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12913268/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146211768","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":"19F-NMR spectroscopy of fluorinated isoleucine analogues in a protein","authors":"Adarshi P. Welegedara,&nbsp;Yi Jiun Tan,&nbsp;Matteo Borgini,&nbsp;Peter Wipf,&nbsp;Gottfried Otting","doi":"10.1007/s10858-026-00488-z","DOIUrl":"10.1007/s10858-026-00488-z","url":null,"abstract":"<div>\u0000 \u0000 <p>The R3H domain of the human protein Sµbp-2 was produced with 5-fluoro-L-isoleucine (FIle) and 5,5-difluoro-L-isoleucine (diFIle) as probes for detection by ¹⁹F-NMR spectroscopy. The fluorinated protein, produced by cell-free protein synthesis, was obtained more easily with diFIle than FIle as FIle readily hydrolysed at pH 7.5 with the release of fluoride. The ¹⁹F-NMR spectra showed large chemical shift ranges but were heterogeneous. The heterogeneities arose from difficulties to fully exclude canonical isoleucine, the presence of multiple conformations and limited stability of the proteins, with the sample made with diFIle being particularly prone to precipitation. ¹⁹F resonance assignments were obtained by comparison of the chemical shifts of γ₁-protons with those observed in the wild-type protein. Non-uniform cross-peak intensities observed in short-delay ¹H,¹⁹F correlation experiments suggest incomplete averaging of ³<i>J</i>_HF couplings and therefore preferential rotamer populations of the CH₂F and CHF₂ groups.</p>\u0000 </div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12913250/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146211754","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":"Web server DDfit: a new scheme to process PFG NMR diffusion data with improved precision","authors":"Vladislav A. Salikov,&nbsp;Olga O. Lebedenko,&nbsp;Nikolai R. Skrynnikov,&nbsp;Ivan S. Podkorytov","doi":"10.1007/s10858-026-00487-0","DOIUrl":"10.1007/s10858-026-00487-0","url":null,"abstract":"<div><p>In this communication we describe a new scheme to process the data from stimulated echo protein diffusion experiments. For a series of gradient-encoded proton spectra <span>(:{f}_{k}left(omega:right))</span> considered over the selected spectral region <span>(:({omega:}_{left},{omega:}_{right}))</span>, we build a model to approximate the unique (protein-dependent) shape of the spectrum. Taking a cue from the optimal filtration theory, <span>(:{f}_{model}left(omega:right))</span> is constructed as the intensity-weighted combination of <span>(:{f}_{k}left(omega:right))</span>. The so obtained <span>(:{f}_{model}left(omega:right))</span> is then used to fit the individual spectra <span>(:{f}_{k}left(omega:right))</span>, thus providing highly accurate estimates for the integral signal intensities that are subsequently used for Stejskal-Tanner-type analyses. This algorithm has been implemented as a part of a new web server, named DDfit (https://ddfit.org, mirror at https://ddfit.bio-nmr.spbu.ru/). The server accepts spectrometer data from the standard stimulated and double-stimulated echo experiments by Bruker, as well as custom-designed experiments. The server is easy to use, with data processing taking no more than several seconds. Our tests using simulated as well as experimental data found that DDfit determines protein diffusion coefficients with both accuracy and precision, offering several-fold improvement in precision compared to other processing schemes.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"80 1","pages":""},"PeriodicalIF":1.9,"publicationDate":"2026-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146211793","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}