Journal of Biomolecular NMR最新文献

{"title":"Measuring long-range contacts in a fully protonated protein at 105 kHz magic angle spinning.","authors":"Zainab O Mustapha, Eren H Ozturk, Benjamin E Lefkin, Diana Grajeda, Andrew J Nieuwkoop","doi":"10.1007/s10858-025-00477-8","DOIUrl":"https://doi.org/10.1007/s10858-025-00477-8","url":null,"abstract":"<p><p>The use of ¹H detection, made possible by very fast magic-angle spinning (MAS), has revolutionized the field of biomolecular solid-state NMR. In the past, ¹H detection was often paired with deuteration schemes to achieve the highest possible resolution needed for protein structural characterization. However, with modern probes capable of MAS rates over 100 kHz, deuteration is no longer required, resulting in a need to measure long-range distances in fully protonated systems. In this study, we evaluate the potential of two 3D pulse sequences, (H)NCOH and (H)NCAH, to measure long-range C-H correlations in a fully protonated protein sample at a MAS rate of 105 kHz. Our results show that the (H)NCOH spectrum contains multiple sequential and structurally relevant long-range CO-H contacts for each residue, capturing H^N contacts up to 6 Å despite transfers to side chain protons. Conversely, the (H)NCAH spectrum yields fewer Cα-H^N correlations, with those present mostly from intraresidue aliphatic proton contacts. Therefore, in protonated proteins, the extensive ¹H network leads to dipolar truncation in the Cα-H experiment, while the CO-H correlations observed are comparable to those in deuterated samples. These findings highlight the feasibility of conducting distance measurements based on long-range cross polarization, on more accessible and affordable samples, expanding the scope of proton detection for systems where deuteration and back-exchange are not possible.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.9,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145147353","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":"Analyzing sub-millisecond timescale protein dynamics using eCPMG experiments.","authors":"Apurva Phale, Aishani Tewari, Gayatri Tendulkar, Ranabir Das, Sivakumar Srinivasan, Kalyan S Chakrabarti","doi":"10.1007/s10858-025-00475-w","DOIUrl":"https://doi.org/10.1007/s10858-025-00475-w","url":null,"abstract":"<p><p>Cellular functions require biomolecules to transition among various conformational sub-states in the energy landscape. A mechanistic understanding of cellular functions requires quantitative knowledge of the kinetics, thermodynamics, and structural features of the biomolecules experiencing exchange between several states. High-power Relaxation Dispersion (RD) NMR experiments have proven very effective for such measurements if the exchange occurs in timescales ranging from microseconds to milliseconds. However, scanning the significantly larger kinetic window within the time limit of instrumental availability and sample stability requires careful optimization of experiments. Understanding biomolecular functions at a mechanistic level depends on fitting such experimental data to theoretical models. However, the reliability of the fit parameters depends on the measurement schemes and is sensitive to experimental noise. Here, we benchmark different measurement schemes along with theoretical models for sub-millisecond timescale exchange and determine the robustness of these models in providing information when the measurements contain noise. Our results show that kinetics can be measured reliably from such experiments. The structural features of the exchanging sub-states, encoded in the chemical shift differences between the states, can be fitted, albeit with significant uncertainties. Information about the minor states is difficult to obtain exclusively from the RD data due to large uncertainties and sensitivity to noise.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.9,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145074046","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":"An optimized ¹³C single-quantum CPMG relaxation dispersion experiment for investigating microsecond-to-millisecond timescale dynamics in large proteins.","authors":"Tairan Yuwen, Jiangshu Liu, Zhilian Xia, Youlin Xia, Paolo Rossi, Charalampos G Kalodimos","doi":"10.1007/s10858-025-00474-x","DOIUrl":"https://doi.org/10.1007/s10858-025-00474-x","url":null,"abstract":"<p><p>Biomolecular dynamics in the microsecond-to-millisecond (µs-ms) timescale are linked to various biological functions, such as enzyme catalysis, allosteric regulation, and ligand recognition. In solution state NMR, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are commonly used to probe µs-ms timescale motions, providing detailed kinetic, thermodynamic, and mechanistic information at the atomic level. For investigating conformational dynamics in high-molecular-weight biomolecules, methyl groups serve as ideal probes due to their favorable relaxation properties, and ¹³C CPMG relaxation dispersion is widely employed for characterizing dynamics in selectively ¹³CH₃-labeled samples. However, conventional schemes that apply CPMG pulses with constant phase are susceptible to artifacts arising from off-resonance effects, radiofrequency (RF) field inhomogeneity and pulse imperfections. In this work we present an optimized¹³C single-quantum (SQ) CPMG experiment incorporating the [0013]-phase cycling scheme, and demonstrate its enhanced robustness against various adverse effects. Moreover, the optimized pulse scheme enables finer sampling of CPMG pulsing frequencies and is suited for studying systems with variable J_CH scalar coupling constants, thereby facilitating comprehensive characterization of µs-ms timescale dynamics of biomolecules with increased precision.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.9,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145022559","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":"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":"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":1.9,"publicationDate":"2025-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12427129/pdf/","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":"OA","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,&nbsp;James M. Gruschus,&nbsp;Ana Opina,&nbsp;Olga Vasalatiy,&nbsp;Nico Tjandra","doi":"10.1007/s10858-025-00468-9","DOIUrl":"10.1007/s10858-025-00468-9","url":null,"abstract":"<div><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></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"79 3","pages":"199 - 208"},"PeriodicalIF":1.9,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-025-00468-9.pdf","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":"OA","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,&nbsp;Lucas Siemons,&nbsp;Ulric B. le Paige,&nbsp;Coline Wiame,&nbsp;Florence Cordier,&nbsp;Nicolas Wolff,&nbsp;Guillaume Bouvignies,&nbsp;Philippe Pelupessy,&nbsp;Fabien Ferrage","doi":"10.1007/s10858-025-00464-z","DOIUrl":"10.1007/s10858-025-00464-z","url":null,"abstract":"<div><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></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"79 3","pages":"163 - 170"},"PeriodicalIF":1.9,"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,&nbsp;Leonid S. Brown,&nbsp;Vlad Ladizhansky","doi":"10.1007/s10858-025-00467-w","DOIUrl":"10.1007/s10858-025-00467-w","url":null,"abstract":"<div><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 <i>Anabaena</i> 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></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"79 3","pages":"181 - 197"},"PeriodicalIF":1.9,"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}