Journal of Biomolecular NMR最新文献

{"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, Bartłomiej Banaś, Clemens Kauffmann, Andreas Beier, Daniel Braun, Irene Ceccolini, Wiktor Koźmiński, Robert Konrat, Anna Zawadzka-Kazimierczuk","doi":"10.1007/s10858-025-00458-x","DOIUrl":"https://doi.org/10.1007/s10858-025-00458-x","url":null,"abstract":"<p><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>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Terminal spin labeling of xylotriose strongly affects interactions in the active site of xylanase BcX.","authors":"Mahin Saberi, René Dekkers, Leonardo Passerini, Martina Huber, Mark Overhand, Marcellus Ubbink","doi":"10.1007/s10858-025-00459-w","DOIUrl":"https://doi.org/10.1007/s10858-025-00459-w","url":null,"abstract":"<p><p>Paramagnetic probes provide long-range distance information and report on minor conformations of biomacromolecules. However, it is important to realize that any probe can affect the system of interest. Here, we report on the effects of attaching a small nitroxide spin label [TEMPO, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl] to xylotriose, a substrate of the enzyme xylanase from Bacillus circulans (BcX). BcX has a long and narrow active site cleft accommodating six xylose units and a secondary binding site on its surface. The aim of the study was to probe the interactions of the substrate with the enzyme using paramagnetic relaxation enhancements (PREs). Binding of the substrate to the surface exposed secondary binding site resulted in strong and localized PREs, indicative of well-defined binding. The xylotriose with diamagnetic control tag was still able to bind the active site cleft, though the rate of exchange was reduced relative to that of untagged xylotriose. The substrate with the paramagnetic TEMPO was not able to bind inside the active site cleft. Also, additional interactions on another surface location showed differences between the paramagnetic substrate and the diamagnetic control, despite the minimal chemical differences between TEMPO modified xylotriose and its reduced, diamagnetic counterpart. Our findings underscore the sensitivity of BcX substrate binding to minor substrate modifications. This study serves as a reminder that any probe, including the attachment of a small paramagnetic group, can affect the behavior of the system under investigation. Even the chemical difference between a paramagnetic tag and its diamagnetic control can result in differences in the molecular interactions.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143612853","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":"Improvement in protein HSQC spectra from addition of betaine.","authors":"Finn O'Dea, Aiden J Seargeant, Jessica Hurcum, Rodolpho do Aido-Machado, Michelle L Rowe, Nicola J Baxter, Jon P Waltho, Jon R Sayers, Mike P Williamson","doi":"10.1007/s10858-025-00463-0","DOIUrl":"https://doi.org/10.1007/s10858-025-00463-0","url":null,"abstract":"<p><p>Addition of glycine betaine up to 1 M gave rise to increased intensity for some weak signals in the HSQC spectra of barnase and Plasmodium falciparum flap endonuclease. The signals that were enhanced were low intensity signals, often from amide groups with rapid internal motion (low order parameter). The majority of signals are however somewhat weaker because of the increased viscosity. Addition of betaine is shown to cause a small but significant overall increase in order parameter, no consistent effect on conformational change on the µs-ms timescale, and a reduction in amide exchange rates, by a factor of about 3. The results are consistent with a model in which betaine leads to a reduction in fluctuations within the bulk water, which in turn produces a reduction in internal fluctuations of the protein.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143595989","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":"Exploring the biochemical landscape of bacterial medium with pyruvate as the exclusive carbon source for NMR studies.","authors":"Çağdaş Dağ, Kerem Kahraman","doi":"10.1007/s10858-025-00462-1","DOIUrl":"https://doi.org/10.1007/s10858-025-00462-1","url":null,"abstract":"<p><p>The use of Escherichia coli for recombinant protein production is a cornerstone in structural biology, particularly for nuclear magnetic resonance (NMR) spectroscopy studies. Understanding the metabolic behavior of E. coli under different carbon sources is critical for optimizing isotope labeling strategies, which are essential for protein structure determination by NMR. Recent advancements, such as mixed pyruvate labeling, have enabled improved backbone resonance assignment in large proteins, making selective isotopic labeling strategies more important than ever for NMR studies. In this study, we aimed to investigate the metabolic adaptations of E. coli when grown on pyruvate as the sole carbon source, a common condition used to achieve selective labeling for NMR spectroscopy. Using NMR-based metabolomics, we tracked key metabolic shifts throughout the culture process to better understand how pyruvate metabolism affects protein production and isotopic labeling. Our results reveal that pyruvate is rapidly depleted before IPTG induction, while acetate and lactate accumulate due to overflow metabolism. These byproducts persist after induction, indicating that pyruvate is diverted into waste pathways, which limits its efficient use in isotope incorporation. This metabolic inefficiency presents a challenge for isotopic labeling protocols that rely on pyruvate as a carbon source for NMR studies. Our results highlight the need to fine-tune pyruvate supplementation to improve metabolic efficiency and isotopic labeling, making this study directly relevant to optimizing protocols for NMR studies involving protein structure determination. These insights provide valuable guidance for enhancing the quality and yield of isotopically labeled proteins in NMR spectroscopy.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565629","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":"Counterintuitive method improves yields of isotopically labelled proteins expressed in flask-cultured Escherichia coli.","authors":"Miguel Ángel Treviño","doi":"10.1007/s10858-025-00461-2","DOIUrl":"https://doi.org/10.1007/s10858-025-00461-2","url":null,"abstract":"<p><p>NMR is a powerful tool for the structural and dynamic study of proteins. One of the necessary conditions for the study of these proteins is their isotopic labelling with ¹⁵N and ¹³C. One of the most widely used methods to obtain these labelled proteins is heterologous expression of the proteins in E. coli using ¹³C-D-glucose and ¹⁵NH₄Cl as the sole nutrient sources. In recent years, the price of ¹³C-D-glucose has almost tripled, making it essential to develop labelling methods that are as cost effective as possible. In this work, different parameters were studied to achieve the most rational use of ¹³C-D-glucose, and an optimized method was developed to obtain labelled proteins with high labelling and low ¹³C-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143536321","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":"Local structure propensities in disordered proteins from cross-correlated NMR spin relaxation.","authors":"Daniel Braun, Clemens Kauffmann, Andreas Beier, Irene Ceccolini, Olga O Lebedenko, Nikolai R Skrynnikov, Robert Konrat","doi":"10.1007/s10858-025-00460-3","DOIUrl":"https://doi.org/10.1007/s10858-025-00460-3","url":null,"abstract":"<p><p>Structurally diverse ensembles of intrinsically disordered proteins or regions are difficult to determine, because experimental observables usually report a conformational average. Therefore, in order to infer the underlying distribution, a set of experiments that measure different aspects of the system is necessary. In principle, there exists a set of cross-correlated relaxation (CCR) rates that report on protein backbone geometry in a complementary way. However, CCR rates are hard to interpret, because geometric information is encoded in an ambiguous way and they present themselves as a convolute of both structure and dynamics. Despite these challenges, CCR rates analyzed within a suitable statistical framework are able to identify conformations in structured proteins. In the context of disordered proteins, we find that this approach has to be adjusted to account for local dynamics via including an additional CCR rate. The results of this study show that CCR rates can be used to characterize structure propensities also in disordered proteins. Instead of using an experimental reference structure, we employed computational spectroscopy to calculate CCR rates from molecular dynamics (MD) simulations and subsequently compared the results to conformations as observed directly in the MD trajectory.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514313","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":"Reducing experimental time through spin-lattice relaxation enhancement via dissolved oxygen.","authors":"Louis-Philippe Picard, Dmitry Pichugin, Shuya Kate Huang, Motasem Suleiman, R Scott Prosser","doi":"10.1007/s10858-024-00457-4","DOIUrl":"https://doi.org/10.1007/s10858-024-00457-4","url":null,"abstract":"<p><p>Large proteins and dilute spin systems within a deuterated background are often characterized by long proton (¹H) spin-lattice relaxation times (T₁), which directly impacts the recycle delay and hence, the total experimental time. Dioxygen (O₂) is a well-known paramagnetic species whose short electronic spin-lattice relaxation time (7.5 ps) contributes to effective spin-lattice relaxation of high gamma nuclei. Oxygen's chemical potential and high diffusivity also allows it to access both the protein exterior and much of the (hydrophobic) interior of the protein. Consequently, at O₂ partial pressures of ~ 10 bar, ¹H and ¹⁹F spin-lattice relaxation rates (R₁) typically reach 3-5 Hz (versus rates of 0.7-1.0 Hz without oxygen) with comparable line-broadening in protein NMR spectra. Using fluoroacetate dehalogenase (FAcD) a soluble 35 kDa homodimeric enzyme, a nanodisc-stabilized G protein-coupled receptor (A_2AR), and bovine serum albumin (BSA) as test cases, a 3-fold savings in time was achieved in acquiring ¹H-¹⁵ N HSQC and ¹⁹F NMR spectra, after oxygenation at 9 bar for 24 h. Additional spin-diffusion effects are anticipated to contribute to uniform ¹H spin-lattice relaxation for both solvent-exposed and buried protons, as demonstrated by T₁ relaxation analysis of amides in ¹⁵N-labeled FAcD. Finally, we show that in protein samples dissolved oxygen pre-equilibrated at 9 bar (pO₂) is largely retained in solution at 20° C or lower, using a standard NMR tube for a period of 3-4 days, thus avoiding the use of specialized apparatus or high-pressure NMR tubes in the spectrometer. The convenience of being able to add or remove the quenching species, while avoiding any complex apparatus in the NMR experiment, makes this a practical tool for both ¹⁹F, ¹H-¹³ C, and ¹H-¹⁵ N NMR studies of proteins.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439688","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}