Journal of Biomolecular NMR最新文献

{"title":"Divide-and-conquer strategy for NMR studies of the E. coli γ-clamp loader complex.","authors":"Sam Mahdi, Irina V Semenova, Irina Bezsonova, Penny J Beuning, Dmitry M Korzhnev","doi":"10.1007/s10858-025-00471-0","DOIUrl":"https://doi.org/10.1007/s10858-025-00471-0","url":null,"abstract":"<p><p>The E. coli γ-clamp loader is a 200 kDa pentameric AAA + ATPase comprised of γ, δ and δ' subunits in a 3:1:1 ratio, which opens the ring shaped β-clamp homodimer and loads it onto DNA in a process essential for DNA replication. The clamp loading is initiated by ATP binding, which induces conformational changes in the clamp loader allowing it to bind and open the β-clamp. This is followed by DNA primer-template binding, ATP hydrolysis, and clamp release onto DNA. Despite a wealth of structural and functional data, dynamics and interactions of the γ-clamp loader and the β-clamp underlying elementary steps of this process remain elusive. Here we employed a \"divide-and-conquer\" strategy for the initial NMR characterization of the γ-clamp loader. A new protocol for the clamp loader assembly was proposed allowing selective incorporation of the isotope-labeled δ and δ' subunits for NMR studies. The nearly complete ¹H, ¹⁵N and ¹³C NMR resonance assignments were obtained for the isolated modular domains of the δ and δ' subunits, which facilitated the assignments of the full-length subunits, and side-chain methyl assignments of the subunits in the context of pentameric γ-clamp loader. NMR chemical shift analysis using the random coil index approach revealed increased flexibility in the ATP, DNA, and β-clamp binding interfaces of the isolated subunits, highlighting a potential significance of conformational dynamics for the clamp loading process. The reported clamp loader assembly protocol and resonance assignments enable the detailed NMR studies of protein dynamics and mechanochemistry of the clamp loading cycle.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144688531","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":"Investigating structural and dynamic changes in cellulose due to nanocrystallization.","authors":"Bijay Laxmi Pradhan, Prince Sen, Krishna Kishor Dey, Manasi Ghosh","doi":"10.1007/s10858-025-00472-z","DOIUrl":"https://doi.org/10.1007/s10858-025-00472-z","url":null,"abstract":"<p><p>Cellulose nanocrystals (CNCs) is synthesized from alpha-cellulose by acid hydrolysis method, and formation of nanocrystallization is comprised by using various microscopic and spectroscopic techniques like PXRD, XPS, Raman, FTIR, PL, UV-Vis, DSC, TGA, DLS, SEM, TEM. Nanocrystalline cellulose shows a notably higher photoluminescence (PL) intensity than cellulose, which enhances its ability to absorb and emit visible light. This increase in PL intensity is attributed to a smaller particle size of CNCs, greater surface area, and quantum confinement effects. The higher intensity of the XPS spectrum further supports the larger surface area of CNCs. PXRD and Raman spectroscopy results show that CNCs has a higher crystallinity index than cellulose. Through deconvolution of the ¹³C CP-MAS SSNMR spectrum, we confirmed a significant reduction in the relative abundance of the amorphous region of cellulose (43.61%) to just 4.97% in CNCs. The ¹³C CP-MAS SSNMR spectrum of CNCs, at the C4, C6, C2C3C5 nuclei sites, can be fitted by two distinct lines for both amorphous and crystalline region, indicating the formation of a co-crystal from two nanocrystallites. Despite this, the principal components of the CSA (chemical shift anisotropy) tensor remain unchanged, suggesting similar electronic environments for these two nanocrystallites. The spin-lattice relaxation time and local correlation time of cellulose and CNCs are determined for chemically distinct carbon nuclei residing on D-glucopyranose units. It is noteworthy that the ¹³C spin-lattice relaxation time and ¹³C local correlation time are longer for each chemically distinct nucleus in CNCs compared to cellulose. It can be predicted by observing the NMR relaxometry data that the longer relaxation time in CNCs is due to the enhancement of crystallinity index. Hence, a correlation between the crystallinity index and nuclear spin dynamics can be established by NMR relaxometry measurements. These findings offer significant insights into the intricate structure and dynamic behavior of cellulose and nanocrystalline cellulose (CNCs), crucial for advancing biomimetic material design, which has huge applications across the pharmaceutical, textile, and cosmetics industries.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144641362","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":"Extending the detectable time window of fast protein dynamics using ¹H_N E-CPMG.","authors":"Dwaipayan Mukhopadhyay, Supriya Pratihar, Stefan Becker, Christian Griesinger","doi":"10.1007/s10858-025-00470-1","DOIUrl":"https://doi.org/10.1007/s10858-025-00470-1","url":null,"abstract":"<p><p>Recent advances in high power NMR relaxation dispersion experiments have significantly enhanced our ability to study fast µs timescale motions in proteins, which are crucial for understanding their biological functions. Here, we have extended the detectable time window of such fast dynamics with the development of extreme power ¹H Carr-Purcell-Meiboom-Gill (¹H E-CPMG) experiments targeted at the backbone amide protons (¹H_N). Using this methodology, artifact-free relaxation dispersion profiles can be obtained up to extreme pulsing conditions with minimal setup effort using commonly used standard NMR hardware. We demonstrate the utility of ¹H E-CPMG on human ubiquitin, revealing that the previously reported peptide flip motion influences a larger region of the protein backbone than previously recognized. Additionally, we directly observed a faster dynamic process at residue T09, aligning with previously predicted pincer mode motion. These findings underscore the effectiveness of ¹H E-CPMG in extending the temporal resolution at which biologically relevant fast protein dynamics can be studied.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144525897","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":"Estimating cross-relaxation rates between methyl and neighboring labile proton spins in high molecular weight proteins.","authors":"Vitali Tugarinov, G Marius Clore","doi":"10.1007/s10858-025-00469-8","DOIUrl":"https://doi.org/10.1007/s10858-025-00469-8","url":null,"abstract":"<p><p>We show that water saturation leads to deleterious losses in sensitivity of methyl signals in selectively methyl-[¹³CH₃]-labeled protein samples of high molecular weight proteins dissolved in H₂O. These losses arise from efficient cross-relaxation between methyl protons and proximal labile protons in the protein structure. A phenomenological model for analysis of methyl intensity decay profiles that involves exchange saturation transfer of magnetization from localized proton spins of water to various labile groups in the protein structure that, in turn, efficiently cross-relax with protons of methyl groups, is described. Analysis of methyl intensity decay profiles with this model allows cross-relaxation rates (σ) between methyl and labile protons to be determined and permits identification of methyl sites in close proximity to labile groups in the protein structure.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144141066","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":"Evaluating the use of lanthanide containing dendrimers for solvent paramagnetic relaxation enhancement.","authors":"Westley Pawloski, James M Gruschus, Ana Opina, Olga Vasalatiy, Nico Tjandra","doi":"10.1007/s10858-025-00468-9","DOIUrl":"https://doi.org/10.1007/s10858-025-00468-9","url":null,"abstract":"<p><p>Paramagnetic relaxation enhancement (PRE) is widely used in biomolecular NMR spectroscopy to obtain long-range distance and orientational information for intra- or intermolecular interactions. In contrast to conventional PRE measurements, which require tethering small molecules containing either a radical or paramagnetic ion to specific sites on the target protein, solvent PRE (sPRE) experiments utilize paramagnetic cosolutes to induce a delocalized PRE effect. Compounds developed as contrast agents in magnetic resonance imaging (MRI) applications typically consist of Gd chelated by a small molecule. Coordinating these Gd-containing small molecules to larger and inert scaffolds has been shown to increase the PRE-effect and produce more effective contrast agents in MRI. Inspired by their use as MRI contrast agent, in this work we evaluate the effectiveness of using a functionalized polyamidoamine (PAMAM) dendrimer for sPRE measurements. Using ubiquitin as a model system, we measured the sPRE effect from a generation 5 PAMAM dendrimer (G5-Gd) as a function of temperature and pH and compared to conventional relaxation agents. We also demonstrated the utility of G5-Gd in sPRE studies to monitor changes in the structures of two proteins as they bind their ligands. These studies highlight the attractive properties of these macromolecular relaxation agents in biomolecular sPRE.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143954946","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":"The SOFAST-HMBC-HMQC experiment for pairing geminal methyl groups in valine and leucine side-chains.","authors":"Ana Paula Aguilar Alva, Lucas Siemons, Ulric B le Paige, Coline Wiame, Florence Cordier, Nicolas Wolff, Guillaume Bouvignies, Philippe Pelupessy, Fabien Ferrage","doi":"10.1007/s10858-025-00464-z","DOIUrl":"https://doi.org/10.1007/s10858-025-00464-z","url":null,"abstract":"<p><p>Methyl groups are essential probes for characterising interactions and dynamics in large proteins. HN-based triple-resonance NMR experiments are often too insensitive for methyl assignments, making a NOESY-based approach an efficient strategy. Linking geminal methyl groups in leucine and valine residues is a crucial step in such NOESY-based methyl resonance assignment strategies. This link can be established unambiguously with the 3D-HMBC-HMQC experiment, introduced for large U-[¹²C, ²H] LV-[¹³CH₃]₂-labelled proteins. Here, we introduce the SOFAST variant of the 3D-HMBC-HMQC experiment which provides spectra with fewer artefacts arising from the water signal and a mean increase in signal-to-noise ratio per unit time of 16% compared to the original experiment with an optimised recovery delay.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143794314","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":"Membrane protein structure determination from Paramagnetic Relaxation Enhancement and internuclear distance restraints.","authors":"Raoul F Vaz, Leonid S Brown, Vlad Ladizhansky","doi":"10.1007/s10858-025-00467-w","DOIUrl":"https://doi.org/10.1007/s10858-025-00467-w","url":null,"abstract":"<p><p>Magic angle spinning nuclear magnetic resonance (MAS NMR) is well suited for the determination of protein structure. The key structural information is obtained in the form of spectral cross peaks between spatially close nuclear spins, but assigning these cross peaks unambiguously to unique spin pairs is often a tedious task because of spectral overlap. Here, we use a seven-helical membrane protein Anabaena Sensory Rhodopsin (ASR) as a model system to demonstrate that transverse Paramagnetic Relaxation Enhancements (PRE) extracted from 2D MAS NMR spectra could be used to obtain a protein structural model. Starting with near complete assignments (93%) of ASR residues, TALOS + predicted backbone dihedral angles and secondary structure restraints in the form of backbone hydrogen bonds are combined with PRE-based restraints and used to generate a coarse model. This model is subsequently utilized as a template reference to facilitate automated assignments of highly ambiguous internuclear correlations. The template is used in an iterative cross peak assignment process and is progressively improved through the inclusion of disambiguated restraints, thereby converging to a low root-mean-square-deviation structural model. In addition to improving structure calculation conversion, the inclusion of PREs also improves packing between helices within an alpha-helical bundle.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143741727","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":"COLMARvista: an open source 2D and pseudo-3D NMR spectral processing, visualization, and analysis software in JavaScript.","authors":"Dawei Li, Rafael Brüschweiler","doi":"10.1007/s10858-025-00465-y","DOIUrl":"https://doi.org/10.1007/s10858-025-00465-y","url":null,"abstract":"<p><p>COLMARvista is presented as a new, highly versatile software for the easy and intuitive processing and visual inspection of 2D and pseudo-3D NMR data both for uniformly and non-uniformly sampled datasets. COLMARvista allows fully autonomous processing of spectra, including zero-filling, apodization, water suppression, Fourier transformation, and phase correction. Its full integration with DEEP Picker and Voigt Fitter programs allows the automated deconvolution and reconstruction of the experimental spectra for highly quantitative analysis, from compound concentration determination to the extraction of cross-peak specific relaxation parameters, even for signals affected by significant overlap with other peaks. COLMARvista is based on JavaScript and, hence, it is computer-architecture and operating-system independent including its advanced graphics. It runs on all recent web browsers and does not require a potentially elaborate operating-system dependent installation. COLMARvista may serve as a paradigm also for other software projects to prevent the stockpiling of once powerful legacy software that became frozen in time, thereby ensuring continuing progress of the NMR field and its software for future generations of NMR spectroscopists.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143741723","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":"Quantifying protein-drug lifetimes in human cells by ¹⁹F NMR spectroscopy.","authors":"Wenkai Zhu, Fatema Bhinderwala, Sarah Rambo, Angela M Gronenborn","doi":"10.1007/s10858-025-00466-x","DOIUrl":"https://doi.org/10.1007/s10858-025-00466-x","url":null,"abstract":"<p><p>The cellular environment is a complex and crowded space, with organelles, compartments and multitudes of molecules engaged in intricate networks of communication that modulate binary protein-ligand/protein interactions. As a result, it is becoming increasingly appreciated that evaluations of protein-drug binding should be carried out in the native cellular environment. Here, we present a proof-of-concept study where we measured the lifetime (1/k_off) of a protein-drug complex in human cells by ¹⁹F NMR spectroscopy using fluorinated Cyclophilin A (CypA) bound to Cyclosporine A (CsA). Harnessing the exceptional detection sensitivity of the trifluoromethyl group attached at the para position of Phe60 in CypA, high-quality 2D ¹⁹F-¹⁹F exchange spectra were obtained in cells. Essentially identical k_off values were observed in cells and in vitro, suggesting that the overall impact of the cellular environment on the lifetime of tfmF60 CypA/CsA complex is minimal. Using similar approaches for quantifying protein-drug lifetimes in the native cellular environment paves the way for efficiently screening drug libraries in human cells by ¹⁹F NMR spectroscopy.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699465","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 complete set of cross-correlated relaxation experiments for determining the protein backbone dihedral angles","authors":"Paulina Bartosińska-Marzec,&nbsp;Bartłomiej Banaś,&nbsp;Clemens Kauffmann,&nbsp;Andreas Beier,&nbsp;Daniel Braun,&nbsp;Irene Ceccolini,&nbsp;Wiktor Koźmiński,&nbsp;Robert Konrat,&nbsp;Anna Zawadzka-Kazimierczuk","doi":"10.1007/s10858-025-00458-x","DOIUrl":"10.1007/s10858-025-00458-x","url":null,"abstract":"<div><p>The investigation of structural propensities of proteins is essential for understanding how they function at the molecular level. NMR, offering atomic-scale information, is often the method of choice. One of the available techniques relies on the cross-correlated relaxation (CCR) effect, whose magnitude is related to local spatial conformation. Application of these methods is difficult if the protein under investigation exhibits high mobility, because NMR observables like CCR rates and chemical shifts present themselves as mere averages of an underlying ensemble distribution. Furthermore, relaxation observables are a convolution of structural and dynamical components. Despite these challenges, it is possible to infer the underlying structural ensemble by combining information from several CCR rates with a different geometrical dependence. In this paper, we present a set of eight CCR experiments tailored for proteins of a highly dynamic nature. Analyzed together, they yield a distribution of backbone dihedral angles for each residue of the protein. The experiments were validated on the folded protein ubiquitin using PDB-deposited NMR structures for comparison. Extraordinary peak separation, achieved by evolving four different chemical shifts, allows for the application of this method to intrinsically disordered proteins in future studies.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"79 2","pages":"79 - 98"},"PeriodicalIF":1.3,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-025-00458-x.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143668537","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}