Journal of Biomolecular NMR最新文献

{"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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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":"<div><p>Over the last decade amide ¹⁵N 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⁻¹), 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’ <i>B</i>_<i>1</i> fields. The ¹⁵N CEST experiment is very sensitive to exchange as the exchange delay <i>T</i>_<i>EX</i> 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 (<span>({p}_{minor})</span>) to as low as 1%. When systems are in fast exchange and the ¹⁵N CEST data has to be described using a model that contains exchange, the exchange parameters are often poorly defined because the <span>({chi }_{red}^{2})</span> versus <span>({p}_{minor})</span> and <span>({chi }_{red}^{2})</span> versus exchange rate (<span>({k}_{ex})</span>) plots can be quite flat with shallow or no minima and the analysis of such ¹⁵N 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 ¹⁵N CEST data acquired with moderate <i>B</i>_<i>1</i> values (~50 to ~350 Hz) results in convincing minima in the <span>({chi }_{red}^{2})</span> versus <span>({p}_{minor})</span> and the <span>({chi }_{red}^{2})</span> versus <span>({k}_{ex})</span> plots even when exchange occurs on the 100 μs timescale. The utility of this strategy is demonstrated on the fast-folding <i>Bacillus stearothermophilus</i> peripheral subunit binding domain that folds with a rate constant ~10⁴ s⁻¹. Here the analysis of ¹⁵N CEST data alone results in <span>({chi }_{red}^{2})</span> versus <span>({p}_{minor})</span> and <span>({chi }_{red}^{2})</span> versus <span>({k}_{ex})</span> 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 ¹⁵N CEST data results in pronounced minima in the <span>({chi }_{red}^{2})</span> versus <span>({p}_{minor})</span> and <span>({chi }_{red}^{2})</span> versus <span>({k}_{ex})</span> plots and precise exchange parameters even in the fast exchange regime (<span>({k}_{ex}/|mathrm{Delta omega }|)</span>~5). Using this strategy we find that the folding rate constant of PSBD is invariant (~10,500 s⁻¹) from 33.2 to 42.9 °C while the unfolding r","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"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":null,"pages":null},"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}

{"title":"Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils","authors":"Alons Lends,&nbsp;Nicolas Birlirakis,&nbsp;Xinyi Cai,&nbsp;Asen Daskalov,&nbsp;Jayakrishna Shenoy,&nbsp;Muhammed Bilal Abdul-Shukkoor,&nbsp;Mélanie Berbon,&nbsp;Fabien Ferrage,&nbsp;Yangping Liu,&nbsp;Antoine Loquet,&nbsp;Kong Ooi Tan","doi":"10.1007/s10858-023-00416-5","DOIUrl":"10.1007/s10858-023-00416-5","url":null,"abstract":"<div><p>Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-β arrangement rich in β-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those of flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (≥ 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of ε ~ 50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε ~ 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4348719","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":"Non-uniform sampling of similar NMR spectra and its application to studies of the interaction between alpha-synuclein and liposomes","authors":"Alexandra Shchukina,&nbsp;Thomas C. Schwarz,&nbsp;Michał Nowakowski,&nbsp;Robert Konrat,&nbsp;Krzysztof Kazimierczuk","doi":"10.1007/s10858-023-00418-3","DOIUrl":"10.1007/s10858-023-00418-3","url":null,"abstract":"<div><p>The accelerated acquisition of multidimensional NMR spectra using sparse non-uniform sampling (NUS) has been widely adopted in recent years. The key concept in NUS is that a major part of the data is omitted during measurement, and then reconstructed using, for example, compressed sensing (CS) methods. CS requires spectra to be compressible, that is, they should contain relatively few “significant” points. The more compressible the spectrum, the fewer experimental NUS points needed in order for it to be accurately reconstructed. In this paper we show that the CS processing of similar spectra can be enhanced by reconstructing only the differences between them. Accurate reconstruction can be obtained at lower sampling levels as the difference is sparser than the spectrum itself. In many situations this method is superior to “conventional” compressed sensing. We exemplify the concept of “difference CS” with one such case—the study of alpha-synuclein binding to liposomes and its dependence on temperature. To obtain information on temperature-dependent transitions between different states, we need to acquire several dozen spectra at various temperatures, with and without the presence of liposomes. Our detailed investigation reveals that changes in the binding modes of the alpha-synuclein ensemble are not only temperature-dependent but also show non-linear behavior in their transitions. Our proposed CS processing approach dramatically reduces the number of NUS points required and thus significantly shortens the experimental time.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-023-00418-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5020485","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":"A methyl-TROSY based 13C relaxation dispersion NMR experiment for studies of chemical exchange in proteins","authors":"Vitali Tugarinov,&nbsp;James L. Baber,&nbsp;G. Marius Clore","doi":"10.1007/s10858-023-00413-8","DOIUrl":"10.1007/s10858-023-00413-8","url":null,"abstract":"<div><p>A methyl Transverse Relaxation Optimized Spectroscopy (methyl-TROSY) based, multiple quantum (MQ) ¹³C Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiment is described. The experiment is derived from the previously developed MQ ¹³C–¹H CPMG scheme (Korzhnev in J Am Chem Soc 126: 3964–73, 2004) supplemented with a CPMG train of refocusing ¹H pulses applied with constant frequency and synchronized with the ¹³C CPMG pulse train. The optimal ¹H ‘decoupling’ scheme that minimizes the amount of fast-relaxing methyl MQ magnetization present during CPMG intervals, makes use of an XY-4 phase cycling of the refocusing composite ¹H pulses. For small-to-medium sized proteins, the MQ ¹³C CPMG experiment has the advantage over its single quantum (SQ) ¹³C counterpart of significantly reducing intrinsic, exchange-free relaxation rates of methyl coherences. For high molecular weight proteins, the MQ ¹³C CPMG experiment eliminates complications in the interpretation of MQ ¹³C–¹H CPMG relaxation dispersion profiles arising from contributions to exchange from differences in methyl ¹H chemical shifts between ground and excited states. The MQ ¹³C CPMG experiment is tested on two protein systems: (1) a triple mutant of the Fyn SH3 domain that interconverts slowly on the chemical shift time scale between the major folded state and an excited state folding intermediate; and (2) the 82-kDa enzyme Malate Synthase G (MSG), where chemical exchange at individual Ile δ1 methyl positions occurs on a much faster time-scale.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-023-00413-8.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5316525","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":"NMR detection and conformational dependence of two, three, and four-bond isotope shifts due to deuteration of backbone amides","authors":"Andrei T. Alexandrescu,&nbsp;Aurelio J. Dregni,&nbsp;Carolyn M. Teschke","doi":"10.1007/s10858-023-00414-7","DOIUrl":"10.1007/s10858-023-00414-7","url":null,"abstract":"<div><p>NMR isotope shifts occur due to small differences in nuclear shielding when nearby atoms are different isotopes. For molecules dissolved in 1:1 H₂O:D₂O, the resulting mixture of N-H and N-D isotopes leads to a small splitting of resonances from adjacent nuclei. We used multidimensional NMR to measure isotope shifts for the proteins CUS-3iD and CspA. We observed four-bond ⁴∆N(ND) isotope shifts in high-resolution 2D ¹⁵N-TROSY experiments of the perdeuterated proteins that correlate with the torsional angle psi. Three-bond ³∆C’(ND) isotope shifts detected in H(N)CO spectra correlate with the intraresidue H-O distance, and to a lesser extent with the dihedral angle phi. The conformational dependence of the isotope shifts agree with those previously reported in the literature. Both the ⁴∆N(ND) and ³∆C’(ND) isotope shifts are sensitive to distances between the atoms giving rise to the isotope shifts and the atoms experiencing the splitting, however, these distances are strongly correlated with backbone dihedral angles making it difficult to resolve distance from stereochemical contributions to the isotope shift. H(NCA)CO spectra were used to measure two-bond ²∆C’(ND) isotope shifts and [D]/[H] fractionation factors. Neither parameter showed significant differences for hydrogen-bonded sites, or changes over a 25° temperature range, suggesting they are not sensitive to hydrogen bonding. Finally, the quartet that arises from the combination of ²∆C’(ND) and ³∆C’(ND) isotope shifts in H(CA)CO spectra was used to measure synchronized hydrogen exchange for the sequence neighbors A315-S316 in the protein CUS-3iD. In many of our experiments we observed minor resonances due to the 10% D₂O used for the sample deuterium lock, indicating isotope shifts can be a source of spectral heterogeneity in standard NMR experiments. We suggest that applications of isotope shifts such as conformational analysis and correlated hydrogen exchange could benefit from the larger magnetic fields becoming available.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4919257","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":"Sparse pseudocontact shift NMR data obtained from a non-canonical amino acid-linked lanthanide tag improves integral membrane protein structure prediction","authors":"Kaitlyn V. Ledwitch,&nbsp;Georg Künze,&nbsp;Jacob R. McKinney,&nbsp;Elleansar Okwei,&nbsp;Katherine Larochelle,&nbsp;Lisa Pankewitz,&nbsp;Soumya Ganguly,&nbsp;Heather L. Darling,&nbsp;Irene Coin,&nbsp;Jens Meiler","doi":"10.1007/s10858-023-00412-9","DOIUrl":"10.1007/s10858-023-00412-9","url":null,"abstract":"<div><p>A single experimental method alone often fails to provide the resolution, accuracy, and coverage needed to model integral membrane proteins (IMPs). Integrating computation with experimental data is a powerful approach to supplement missing structural information with atomic detail. We combine RosettaNMR with experimentally-derived paramagnetic NMR restraints to guide membrane protein structure prediction. We demonstrate this approach using the disulfide bond formation protein B (DsbB), an α-helical IMP. Here, we attached a cyclen-based paramagnetic lanthanide tag to an engineered non-canonical amino acid (ncAA) using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction. Using this tagging strategy, we collected 203 backbone H^N pseudocontact shifts (PCSs) for three different labeling sites and used these as input to guide <i>de novo</i> membrane protein structure prediction protocols in Rosetta. We find that this sparse PCS dataset combined with 44 long-range NOEs as restraints in our calculations improves structure prediction of DsbB by enhancements in model accuracy, sampling, and scoring. The inclusion of this PCS dataset improved the Cα-RMSD transmembrane segment values of the best-scoring and best-RMSD models from 9.57 Å and 3.06 Å (no NMR data) to 5.73 Å and 2.18 Å, respectively.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-023-00412-9.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4490545","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}