Journal of Biomolecular NMR最新文献

{"title":"5D solid-state NMR spectroscopy for facilitated resonance assignment","authors":"Alexander Klein,&nbsp;Suresh K. Vasa,&nbsp;Rasmus Linser","doi":"10.1007/s10858-023-00424-5","DOIUrl":"10.1007/s10858-023-00424-5","url":null,"abstract":"<div><p>¹H-detected solid-state NMR spectroscopy has been becoming increasingly popular for the characterization of protein structure, dynamics, and function. Recently, we showed that higher-dimensionality solid-state NMR spectroscopy can aid resonance assignments in large micro-crystalline protein targets to combat ambiguity (Klein et al., Proc. Natl. Acad. Sci. U.S.A. 2022). However, assignments represent both, a time-limiting factor and one of the major practical disadvantages within solid-state NMR studies compared to other structural-biology techniques from a very general perspective. Here, we show that 5D solid-state NMR spectroscopy is not only justified for high-molecular-weight targets but will also be a realistic and practicable method to streamline resonance assignment in small to medium-sized protein targets, which such methodology might not have been expected to be of advantage for. Using a combination of non-uniform sampling and the signal separating algorithm for spectral reconstruction on a deuterated and proton back-exchanged micro-crystalline protein at fast magic-angle spinning, direct amide-to-amide correlations in five dimensions are obtained with competitive sensitivity compatible with common hardware and measurement time commitments. The self-sufficient backbone walks enable efficient assignment with very high confidence and can be combined with higher-dimensionality sidechain-to-backbone correlations from protonated preparations into minimal sets of experiments to be acquired for simultaneous backbone and sidechain assignment. The strategies present themselves as potent alternatives for efficient assignment compared to the traditional assignment approaches in 3D, avoiding user misassignments derived from ambiguity or loss of overview and facilitating automation. This will ease future access to NMR-based characterization for the typical solid-state NMR targets at fast MAS.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 5-6","pages":"229 - 245"},"PeriodicalIF":2.7,"publicationDate":"2023-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10687145/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71520147","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":"Paramagnetic NMR to study iron sulfur proteins: 13C detected experiments illuminate the vicinity of the metal center","authors":"Leonardo Querci,&nbsp;Deborah Grifagni,&nbsp;Inês B. Trindade,&nbsp;José Malanho Silva,&nbsp;Ricardo O. Louro,&nbsp;Francesca Cantini,&nbsp;Mario Piccioli","doi":"10.1007/s10858-023-00425-4","DOIUrl":"10.1007/s10858-023-00425-4","url":null,"abstract":"<div><p>The robustness of NMR coherence transfer in proximity of a paramagnetic center depends on the relaxation properties of the nuclei involved. In the case of Iron-Sulfur Proteins, different pulse schemes or different parameter sets often provide complementary results. Tailored versions of HCACO and CACO experiments significantly increase the number of observed C^α/C’ connectivities in highly paramagnetic systems, by recovering many resonances that were lost due to paramagnetic relaxation. Optimized ¹³C direct detected experiments can significantly extend the available assignments, improving the overall knowledge of these systems. The different relaxation properties of C^α and C’ nuclei are exploited in CACO vs COCA experiments and the complementarity of the two experiments is used to obtain structural information. The two [Fe₂S₂]⁺ clusters containing NEET protein CISD3 and the one [Fe₄S₄]²⁺ cluster containing HiPIP protein PioC have been taken as model systems. We show that tailored experiments contribute to decrease the blind sphere around the cluster, to extend resonance assignment of cluster bound cysteine residues and to retrieve details on the topology of the iron-bound ligand residues.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 5-6","pages":"247 - 259"},"PeriodicalIF":2.7,"publicationDate":"2023-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10687126/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49673020","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":"Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics","authors":"Theresa Höfurthner,&nbsp;Giorgia Toscano,&nbsp;Georg Kontaxis,&nbsp;Andreas Beier,&nbsp;Moriz Mayer,&nbsp;Leonhard Geist,&nbsp;Darryl B. McConnell,&nbsp;Harald Weinstabl,&nbsp;Roman Lichtenecker,&nbsp;Robert Konrat","doi":"10.1007/s10858-023-00427-2","DOIUrl":"10.1007/s10858-023-00427-2","url":null,"abstract":"<div><p>In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum ¹³C−¹ H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased ¹³C-T₂ times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 1","pages":"1 - 8"},"PeriodicalIF":1.3,"publicationDate":"2023-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10981609/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41187718","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":"Breaking boundaries: TINTO in POKY for computer vision-based NMR walking strategies","authors":"Andrea Estefania Lopez Giraldo,&nbsp;Zowie Werner,&nbsp;Mehdi Rahimi,&nbsp;Woonghee Lee","doi":"10.1007/s10858-023-00423-6","DOIUrl":"10.1007/s10858-023-00423-6","url":null,"abstract":"<div><p>Nuclear magnetic resonance is a crucial technique for studying biological complexes, as it provides precise structural and dynamic information at the atomic level. However, the process of assigning resonances can be time-consuming and challenging, particularly in cases where peaks overlap, or the data quality is poor. In this paper, we present TINTO (Two and three-dimensional Imaging for NMR sTrip Operation via CV/ML), an advanced semiautomatic toolset for NMR resonance assignment. TINTO comprises two separate tools, each tailored for either two-dimensional or three-dimensional imaging. The toolset utilizes a computer-vision approach and a machine learning approach, specifically structural similarity index and principal components analysis, to perform visual similarity searches of resonances and quickly locate similar strips, and in that way overcome the challenges associated with peak overlap without requiring peak picking. Our tool offers a user-friendly interface and has the potential to enhance the efficiency and accuracy of NMR resonance assignment, particularly in complex cases. This advancement holds promising implications for furthering our understanding of biological systems at the molecular level. TINTO is pre-installed in the POKY suite, which is available at https://poky.clas.ucdenver.edu.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 5-6","pages":"217 - 228"},"PeriodicalIF":2.7,"publicationDate":"2023-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41113412","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 thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins","authors":"Matej Dzurov,&nbsp;Šárka Pospíšilová,&nbsp;Michaela Krafčíková,&nbsp;Lukáš Trantírek,&nbsp;Lucy Vojtová,&nbsp;Jan Ryneš","doi":"10.1007/s10858-023-00422-7","DOIUrl":"10.1007/s10858-023-00422-7","url":null,"abstract":"<div><p>Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts of their metabolism. The main drawbacks of commonly used matrices include the inability to recover intact cells post-measurement for additional analyses and/or requirements for specific operating temperatures. Here, we report on the development and characterization of a set of thermosensitive and nontoxic triblock copolymers based on poly(D,L-lactide)-<i>b</i>-poly(ethylene glycol)-<i>b</i>-poly(D,L-lactide) (PLA-PEG-PLA). Here, we show for the first time that these copolymers are suitable as immobilizer matrices for the acquisition of in-cell NMR spectra of nucleic acids and proteins over a commonly used sample temperature range of 15–40 °C and, importantly, allow recovery of cells after completion of in-cell NMR spectra acquisition. We compared the performances of currently used matrices in terms of cell viability (dye exclusion assays), cellular metabolism (1D ³¹P NMR), and quality of in-cell NMR spectra of two model biomacromolecules (hybrid double-stranded/i-motif DNA and ubiquitin). Our results demonstrate the suitability and advantages of PLA-PEG-PLA copolymers for application in bioreactor-assisted in-cell NMR.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 5-6","pages":"203 - 215"},"PeriodicalIF":2.7,"publicationDate":"2023-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10687187/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10189696","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":"Strategies for acquisition of resonance assignment spectra of highly dynamic membrane proteins: a GPCR case study","authors":"Evan J. van Aalst,&nbsp;Jun Jang,&nbsp;Ty C. Halligan,&nbsp;Benjamin J. Wylie","doi":"10.1007/s10858-023-00421-8","DOIUrl":"10.1007/s10858-023-00421-8","url":null,"abstract":"<div><p>In protein nuclear magnetic resonance (NMR), chemical shift assignment provides a wealth of information. However, acquisition of high-quality solid-state NMR spectra depends on protein-specific dynamics. For membrane proteins, bilayer heterogeneity further complicates this observation. Since the efficiency of cross-polarization transfer is strongly entwined with protein dynamics, optimal temperatures for spectral sensitivity and resolution will depend not only on inherent protein dynamics, but temperature-dependent phase properties of the bilayer environment. We acquired 1-, 2-, and 3D homo- and heteronuclear experiments of the chemokine receptor CCR3 in a 7:3 phosphatidylcholine:cholesterol lipid environment. 1D direct polarization, cross polarization (CP), and T₂’ experiments indicate sample temperatures below − 25 °C facilitate higher CP enhancement and longer-lived transverse relaxation times. T₁_rho experiments indicate intermediate timescales are minimized below a sample temperature of − 20 °C. 2D DCP NCA experiments indicated optimal CP efficiency and resolution at a sample temperature of − 30 °C, corroborated by linewidth analysis in 3D NCACX at − 30 °C compared to − 5 °C. This optimal temperature is concluded to be directly related the lipid phase transition, measured to be between − 20 and 15 °C based on rINEPT signal of all-trans and trans-gauche lipid acyl conformations. Our results have critical implications in acquisition of SSNMR membrane protein assignment spectra, as we hypothesize that different lipid compositions with different phase transition properties influence protein dynamics and therefore the optimal acquisition temperature.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 4","pages":"191 - 202"},"PeriodicalIF":2.7,"publicationDate":"2023-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5411758","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":"Improved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors","authors":"Damian Van Raad,&nbsp;Thomas Huber,&nbsp;Gottfried Otting","doi":"10.1007/s10858-023-00420-9","DOIUrl":"10.1007/s10858-023-00420-9","url":null,"abstract":"<div><p>Cell-free protein synthesis using eCells allows production of amino acids from inexpensive ¹³C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of ¹³C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [¹³C,¹H]-HSQC cross-peaks free of one-bond ¹³C–¹³C couplings. Selective ¹³C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 4","pages":"183 - 190"},"PeriodicalIF":2.7,"publicationDate":"2023-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-023-00420-9.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5093205","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":"E. coli “Stablelabel” S30 lysate for optimized cell-free NMR sample preparation","authors":"Roman Levin,&nbsp;Frank Löhr,&nbsp;Betül Karakoc,&nbsp;Roman Lichtenecker,&nbsp;Volker Dötsch,&nbsp;Frank Bernhard","doi":"10.1007/s10858-023-00417-4","DOIUrl":"10.1007/s10858-023-00417-4","url":null,"abstract":"<div><p>Cell-free (CF) synthesis with highly productive <i>E. coli</i> lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of ¹⁵N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized <i>E. coli</i> lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the <i>E. coli</i> strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative “Stablelabel” containing the cumulative mutations <i>asnA, ansA/B, glnA, aspC</i> and <i>ilvE</i> yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in “Stablelabel” lysates. By taking advantage of <i>ilvE</i> deletion in \"Stablelabel\", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 4","pages":"131 - 147"},"PeriodicalIF":2.7,"publicationDate":"2023-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-023-00417-4.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4545957","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":"Studying micro to millisecond protein dynamics using simple amide 15N CEST experiments supplemented with major-state R2 and visible peak-position constraints","authors":"Nihar Pradeep Khandave,&nbsp;Ashok Sekhar,&nbsp;Pramodh Vallurupalli","doi":"10.1007/s10858-023-00419-2","DOIUrl":"10.1007/s10858-023-00419-2","url":null,"abstract":"&lt;div&gt;&lt;p&gt;Over the last decade amide &lt;sup&gt;15&lt;/sup&gt;N CEST experiments have emerged as a popular tool to study protein dynamics that involves exchange between a ‘visible’ major state and sparsely populated ‘invisible’ minor states. Although initially introduced to study exchange between states that are in slow exchange with each other (typical exchange rates of, 10 to 400 s&lt;sup&gt;−1&lt;/sup&gt;), they are now used to study interconversion between states on the intermediate to fast exchange timescale while still using low to moderate (5 to 350 Hz) ‘saturating’ &lt;i&gt;B&lt;/i&gt;&lt;sub&gt;&lt;i&gt;1&lt;/i&gt;&lt;/sub&gt; fields. The &lt;sup&gt;15&lt;/sup&gt;N CEST experiment is very sensitive to exchange as the exchange delay &lt;i&gt;T&lt;/i&gt;&lt;sub&gt;&lt;i&gt;EX&lt;/i&gt;&lt;/sub&gt; can be quite long (~0.5 s) allowing for a large number of exchange events to occur making it a very powerful tool to detect minor sates populated (&lt;span&gt;({p}_{minor})&lt;/span&gt;) to as low as 1%. When systems are in fast exchange and the &lt;sup&gt;15&lt;/sup&gt;N CEST data has to be described using a model that contains exchange, the exchange parameters are often poorly defined because the &lt;span&gt;({chi }_{red}^{2})&lt;/span&gt; versus &lt;span&gt;({p}_{minor})&lt;/span&gt; and &lt;span&gt;({chi }_{red}^{2})&lt;/span&gt; versus exchange rate (&lt;span&gt;({k}_{ex})&lt;/span&gt;) plots can be quite flat with shallow or no minima and the analysis of such &lt;sup&gt;15&lt;/sup&gt;N CEST data can lead to wrong estimates of the exchange parameters due to the presence of ‘spurious’ minima. Here we show that the inclusion of experimentally derived constraints on the intrinsic transverse relaxation rates and the inclusion of visible state peak-positions during the analysis of amide &lt;sup&gt;15&lt;/sup&gt;N CEST data acquired with moderate &lt;i&gt;B&lt;/i&gt;&lt;sub&gt;&lt;i&gt;1&lt;/i&gt;&lt;/sub&gt; values (~50 to ~350 Hz) results in convincing minima in the &lt;span&gt;({chi }_{red}^{2})&lt;/span&gt; versus &lt;span&gt;({p}_{minor})&lt;/span&gt; and the &lt;span&gt;({chi }_{red}^{2})&lt;/span&gt; versus &lt;span&gt;({k}_{ex})&lt;/span&gt; plots even when exchange occurs on the 100 μs timescale. The utility of this strategy is demonstrated on the fast-folding &lt;i&gt;Bacillus stearothermophilus&lt;/i&gt; peripheral subunit binding domain that folds with a rate constant ~10&lt;sup&gt;4&lt;/sup&gt; s&lt;sup&gt;−1&lt;/sup&gt;. Here the analysis of &lt;sup&gt;15&lt;/sup&gt;N CEST data alone results in &lt;span&gt;({chi }_{red}^{2})&lt;/span&gt; versus &lt;span&gt;({p}_{minor})&lt;/span&gt; and &lt;span&gt;({chi }_{red}^{2})&lt;/span&gt; versus &lt;span&gt;({k}_{ex})&lt;/span&gt; plots that contain shallow minima, but the inclusion of visible-state peak positions and restraints on the intrinsic transverse relaxation rates of both states during the analysis of the &lt;sup&gt;15&lt;/sup&gt;N CEST data results in pronounced minima in the &lt;span&gt;({chi }_{red}^{2})&lt;/span&gt; versus &lt;span&gt;({p}_{minor})&lt;/span&gt; and &lt;span&gt;({chi }_{red}^{2})&lt;/span&gt; versus &lt;span&gt;({k}_{ex})&lt;/span&gt; plots and precise exchange parameters even in the fast exchange regime (&lt;span&gt;({k}_{ex}/|mathrm{Delta omega }|)&lt;/span&gt;~5). Using this strategy we find that the folding rate constant of PSBD is invariant (~10,500 s&lt;sup&gt;−1&lt;/sup&gt;) from 33.2 to 42.9 °C while the unfolding r","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 4","pages":"165 - 181"},"PeriodicalIF":2.7,"publicationDate":"2023-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4426356","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":"1H-detected characterization of carbon–carbon networks in highly flexible protonated biomolecules using MAS NMR","authors":"Salima Bahri,&nbsp;Adil Safeer,&nbsp;Agnes Adler,&nbsp;Hanneke Smedes,&nbsp;Hugo van Ingen,&nbsp;Marc Baldus","doi":"10.1007/s10858-023-00415-6","DOIUrl":"10.1007/s10858-023-00415-6","url":null,"abstract":"<div><p>In the last three decades, the scope of solid-state NMR has expanded to exploring complex biomolecules, from large protein assemblies to intact cells at atomic-level resolution. This diversity in macromolecules frequently features highly flexible components whose insoluble environment precludes the use of solution NMR to study their structure and interactions. While High-resolution Magic-Angle Spinning (HR-MAS) probes offer the capacity for gradient-based ¹H-detected spectroscopy in solids, such probes are not commonly used for routine MAS NMR experiments. As a result, most exploration of the flexible regime entails either ¹³C-detected experiments, the use of partially perdeuterated systems, or ultra-fast MAS. Here we explore proton-detected pulse schemes probing through-bond ¹³C–¹³C networks to study mobile protein sidechains as well as polysaccharides in a broadband manner. We demonstrate the use of such schemes to study a mixture of microtubule-associated protein (MAP) tau and human microtubules (MTs), and the cell wall of the fungus <i>Schizophyllum commune</i> using 2D and 3D spectroscopy, to show its viability for obtaining unambiguous correlations using standard fast-spinning MAS probes at high and ultra-high magnetic fields.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 3","pages":"111 - 119"},"PeriodicalIF":2.7,"publicationDate":"2023-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-023-00415-6.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4346299","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}