Journal of Biomolecular NMR最新文献

{"title":"1H R1ρ relaxation dispersion experiments in aromatic side chains","authors":"Matthias Dreydoppel,&nbsp;Roman J. Lichtenecker,&nbsp;Mikael Akke,&nbsp;Ulrich Weininger","doi":"10.1007/s10858-021-00382-w","DOIUrl":"10.1007/s10858-021-00382-w","url":null,"abstract":"<div><p>Aromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic ¹H <i>R</i>_1<i>ρ</i> relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated ¹H–¹³C spin pairs with vicinal ²H–¹²C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the ³<i>J</i>_HH couplings and limited chemical shift differences among ¹H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, <i>k</i>_flip, agree well with previous results from ¹³C <i>R</i>_1<i>ρ</i> relaxation dispersion experiments, and yield ¹H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic¹H <i>R</i>_1<i>ρ</i> relaxation dispersion experiment in combination with the site-selective ¹H–¹³C/²H–¹²C labeling scheme enable measurement of exchange rates up to <i>k</i>_ex = 2<i>k</i>_flip = 80,000 s^–1, and serve as a useful complement to previously developed ¹³C-based methods.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00382-w.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4814138","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":"CSI-LSTM: a web server to predict protein secondary structure using bidirectional long short term memory and NMR chemical shifts","authors":"Zhiwei Miao,&nbsp;Qianqian Wang,&nbsp;Xiongjie Xiao,&nbsp;Ghulam Mustafa Kamal,&nbsp;Linhong Song,&nbsp;Xu Zhang,&nbsp;Conggang Li,&nbsp;Xin Zhou,&nbsp;Bin Jiang,&nbsp;Maili Liu","doi":"10.1007/s10858-021-00383-9","DOIUrl":"10.1007/s10858-021-00383-9","url":null,"abstract":"<div><p>Protein secondary structure provides rich structural information, hence the description and understanding of protein structure relies heavily on it. Identification or prediction of secondary structures therefore plays an important role in protein research. In protein NMR studies, it is more convenient to predict secondary structures from chemical shifts as compared to the traditional determination methods based on inter-nuclear distances provided by NOESY experiment. In recent years, there was a significant improvement observed in deep neural networks, which had been applied in many research fields. Here we proposed a deep neural network based on bidirectional long short term memory (biLSTM) to predict protein 3-state secondary structure using NMR chemical shifts of backbone nuclei. While comparing with the existing methods the proposed method showed better prediction accuracy. Based on the proposed method, a web server has been built to provide protein secondary structure prediction service.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4515765","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":"Evaluation of the tert-butyl group as a probe for NMR studies of macromolecular complexes","authors":"Rashmi Voleti,&nbsp;Sofia Bali,&nbsp;Jaime Guerrero,&nbsp;Jared Smothers,&nbsp;Charis Springhower,&nbsp;Gerardo A. Acosta,&nbsp;Kyle D. Brewer,&nbsp;Fernando Albericio,&nbsp;Josep Rizo","doi":"10.1007/s10858-021-00380-y","DOIUrl":"10.1007/s10858-021-00380-y","url":null,"abstract":"<div><p>The development of methyl transverse relaxation optimized spectroscopy has greatly facilitated the study of macromolecular assemblies by solution NMR spectroscopy. However, limited sample solubility and stability has hindered application of this technique to ongoing studies of complexes formed on membranes by the neuronal SNAREs that mediate neurotransmitter release and synaptotagmin-1, the Ca²⁺ sensor that triggers release. Since the ¹H NMR signal of a ^tBu group attached to a large protein or complex can be observed with high sensitivity if the group retains high mobility, we have explored the use of this strategy to analyze presynaptic complexes involved in neurotransmitter release. For this purpose, we attached ^tBu groups at single cysteines of fragments of synaptotagmin-1, complexin-1 and the neuronal SNAREs by reaction with 5-(tert-butyldisulfaneyl)-2-nitrobenzoic acid (BDSNB), ^tBu iodoacetamide or ^tBu acrylate. The ^tBu resonances of the tagged proteins were generally sharp and intense, although ^tBu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the ^tBu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the ^tBu resonances in some cases. However, sharp ^tBu resonances could readily be observed for some complexes of more than 200 kDa at low micromolar concentrations. Our results show that tagging of proteins with ^tBu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00380-y.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4713517","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":"TRACT revisited: an algebraic solution for determining overall rotational correlation times from cross-correlated relaxation rates","authors":"Scott A. Robson,&nbsp;Çağdaş Dağ,&nbsp;Hongwei Wu,&nbsp;Joshua J. Ziarek","doi":"10.1007/s10858-021-00379-5","DOIUrl":"10.1007/s10858-021-00379-5","url":null,"abstract":"<div><p>Accurate rotational correlation times (<span>({tau }_{text{c}})</span>) are critical for quantitative analysis of fast timescale NMR dynamics. As molecular weights increase, the classic derivation of <span>({tau }_{c})</span> using transverse and longitudinal relaxation rates becomes increasingly unsuitable due to the non-trivial contribution of remote dipole–dipole interactions to longitudinal relaxation. Derivations using cross-correlated relaxation experiments, such as TRACT, overcome these limitations but are erroneously calculated in 65% of the citing literature. Herein, we developed an algebraic solutions to the Goldman relationship that facilitate rapid, point-by-point calculations for straightforward identification of appropriate spectral regions where global tumbling is likely to be dominant. The rigid-body approximation of the Goldman relationship has been previously shown to underestimate TRACT-based rotational correlation time estimates. This motivated us to develop a second algebraic solution that employs a simplified model-free spectral density function including an order parameter term that could, in principle, be set to an average backbone S² ≈ 0.9 to further improve the accuracy of <span>({tau }_{text{c}})</span> estimation. These solutions enabled us to explore the boundaries of the Goldman relationship as a function of the H–N internuclear distance (<span>(r)</span>), difference of the two principal components of the axially-symmetric ¹⁵N CSA tensor (<span>(Delta {delta }_{N})</span>), and angle of the CSA tensor relative to the N–H bond vector (<span>(theta)</span>). We hope our algebraic solutions and analytical strategies will increase the accuracy and application of the TRACT experiment.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00379-5.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4143933","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":"Off-resonance 13C–2H REDOR NMR for site-resolved studies of molecular motion","authors":"Martin D. Gelenter,&nbsp;Kelly J. Chen,&nbsp;Mei Hong","doi":"10.1007/s10858-021-00377-7","DOIUrl":"10.1007/s10858-021-00377-7","url":null,"abstract":"<div><p>We introduce a ¹³C–²H Rotational Echo DOuble Resonance (REDOR) technique that uses the difference between on-resonance and off-resonance ²H irradiation to detect dynamic segments in deuterated molecules. By selectively inverting specific regions of the ²H magic-angle spinning (MAS) sideband manifold to recouple some of the deuterons to nearby carbons, we distinguish dynamic and rigid residues in 1D and 2D ¹³C spectra. We demonstrate this approach on deuterated GB1, H/D exchanged GB1, and perdeuterated bacterial cellulose. Numerical simulations reproduce the measured mixing-time and ²H carrier-frequency dependence of the REDOR dephasing of bacterial cellulose. Combining numerical simulations with experiments thus allow the extraction of motionally averaged quadrupolar couplings from REDOR dephasing values.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00377-7.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4110760","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":"An automated iterative approach for protein structure refinement using pseudocontact shifts","authors":"Stefano Cucuzza,&nbsp;Peter Güntert,&nbsp;Andreas Plückthun,&nbsp;Oliver Zerbe","doi":"10.1007/s10858-021-00376-8","DOIUrl":"10.1007/s10858-021-00376-8","url":null,"abstract":"<div><p>NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM₄A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00376-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4071154","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":"Toho-1 β-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam","authors":"Varun V. Sakhrani,&nbsp;Rittik K. Ghosh,&nbsp;Eduardo Hilario,&nbsp;Kevin L. Weiss,&nbsp;Leighton Coates,&nbsp;Leonard J. Mueller","doi":"10.1007/s10858-021-00375-9","DOIUrl":"10.1007/s10858-021-00375-9","url":null,"abstract":"<div><p>Backbone chemical shift assignments for the Toho-1 β-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly ²H,¹³C,¹⁵N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-β-lactam β-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of ¹⁵N relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~ 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00375-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4164701","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":"Antipsychotic phenothiazine drugs bind to KRAS in vitro","authors":"Xu Wang,&nbsp;Alemayehu A. Gorfe,&nbsp;John A. Putkey","doi":"10.1007/s10858-021-00371-z","DOIUrl":"10.1007/s10858-021-00371-z","url":null,"abstract":"<div><p>We used NMR to show that the antipsychotic phenothiazine drugs promazine and promethazine bind to GDP-KRAS. Promazine also binds to oncogenic GDP-KRAS(G12D), and to wild type GppNHp-KRAS. A panel of additional phenothiazines bind to GDP-KRAS but with lower affinity than promazine or promethazine. Binding is most dependent on substitutions at C-2 of the tricyclic phenothiazine ring. Promazine was used to generate an NMR-driven HADDOCK model of the drug/GDP-KRAS complex. The structural model shows the tricyclic phenothiazine ring of promazine associates with the hydrophobic pocket p1 that is bordered by the central β sheet and Switch II in KRAS. Binding appears to stabilize helix 2 in a conformation that is similar to that seen in KRAS bound to other small molecules. Association of phenothiazines with KRAS may affect normal KRAS signaling that could contribute to multiple biological activities of these antipsychotic drugs. Moreover, the phenothiazine ring represents a new core scaffold on which to design modulators of KRAS activity.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00371-z","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5010348","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":"Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution","authors":"Morgane Callon,&nbsp;Alexander A. Malär,&nbsp;Sara Pfister,&nbsp;Václav Římal,&nbsp;Marco E. Weber,&nbsp;Thomas Wiegand,&nbsp;Johannes Zehnder,&nbsp;Matías Chávez,&nbsp;Riccardo Cadalbert,&nbsp;Rajdeep Deb,&nbsp;Alexander Däpp,&nbsp;Marie-Laure Fogeron,&nbsp;Andreas Hunkeler,&nbsp;Lauriane Lecoq,&nbsp;Anahit Torosyan,&nbsp;Dawid Zyla,&nbsp;Rudolf Glockshuber,&nbsp;Stefanie Jonas,&nbsp;Michael Nassal,&nbsp;Matthias Ernst,&nbsp;Anja Böckmann,&nbsp;Beat H. Meier","doi":"10.1007/s10858-021-00373-x","DOIUrl":"10.1007/s10858-021-00373-x","url":null,"abstract":"<div><p>Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in ¹³C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for ¹H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00373-x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4976076","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":"Extensively sparse 13C labeling to simplify solid-state NMR 13C spectra of membrane proteins","authors":"Qiong Tong,&nbsp;Huan Tan,&nbsp;Jianping Li,&nbsp;Huayong Xie,&nbsp;Yongxiang Zhao,&nbsp;Yanke Chen,&nbsp;Jun Yang","doi":"10.1007/s10858-021-00372-y","DOIUrl":"10.1007/s10858-021-00372-y","url":null,"abstract":"<div><p>Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging technique to investigate the structures and dynamics of membrane proteins in an artificial or native membrane environment. However, the structural studies of proteins by ssNMR are usually prolonged or impeded by signal assignments, especially the assignments of signals for collection of distance restraints, because of serious overlapping of signals in 2D ¹³C–¹³C spectra. Sparse labeling of ¹³C spins is an effective approach to simplify the ¹³C spectra and facilitate the extractions of distance restraints. Here, we propose a new reverse labeling combination of six types of amino acid residues (Ile, Leu, Phe, Trp, Tyr and Lys), and show a clean reverse labeling effect on a model membrane protein <i>E. coli</i> aquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate ¹³C–¹²C labeling, and demonstrate an enhanced dilution effect in ¹³C–¹³C spectra. In addition, the influences of reverse labeling on the labeling of the other types of residues are quantitatively analyzed in the two strategies (1, reverse labeling and 2, reverse labeling combining alternate ¹³C–¹²C labeling). The signal intensities of some other types of residues in 2D ¹³C–¹³C spectra are observed to be 20–50% weaker because of the unwanted reverse labeling. The extensively sparse ¹³C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D ¹³C–¹³C spectra of membrane proteins.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00372-y","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4793191","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}