Journal of Biomolecular NMR最新文献

{"title":"Combined multi‐band decoupling in biomolecular NMR spectroscopy","authors":"Clemens Anklin, R. Andrew Byrd","doi":"10.1007/s10858-021-00360-2","DOIUrl":"https://doi.org/10.1007/s10858-021-00360-2","url":null,"abstract":"","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00360-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4871938","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":"Removal of 2H-decoupling sidebands in 13CHD2 13C-CEST profiles","authors":"Youlin Xia,&nbsp;Tairan Yuwen,&nbsp;Aizhuo Liu,&nbsp;Charalampos G. Kalodimos","doi":"10.1007/s10858-021-00362-0","DOIUrl":"https://doi.org/10.1007/s10858-021-00362-0","url":null,"abstract":"<p>A unique aspect of NMR is its capacity to provide integrated insight into both the structure and intrinsic dynamics of biomolecules. Chemical exchange phenomena that often serve as probes of dynamic processes in biological macromolecules can be quantitatively investigated with chemical exchange saturation transfer (CEST) experiments. ²H-decoupling sidebands, however, always occur in the profiles of ¹³CHD₂ ¹³C-CEST experiments when using the simple CW (continuous wave) method, which may obscure the detection of minor dips of excited states. Traditionally, these sidebands are manually eliminated from the profiles before data analysis by removing experimental points in the range of ²H-decoupling field strength?±50?Hz away from the major dips of the ground state on either side of the dips. Unfortunately, this may also eliminate potential minor dips if they overlap with the decoupling sidebands. Here, we developed methods that use pseudo-continuous waves with variable RF amplitudes distributed onto ramps for ²H decoupling. The new methods were thoroughly validated on Bruker spectrometers at a range of fields (¹H frequencies of 600, 700, and 850?MHz, and 1.1 GHz). By using these methods, we successfully removed the sidebands from the NMR profiles of ¹³CHD₂ ¹³C-CEST experiments.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00362-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4802300","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":"Simultaneous measurement of 1HC/N-R2′s for rapid acquisition of backbone and sidechain paramagnetic relaxation enhancements (PREs) in proteins","authors":"C. Ashley Barnes,&nbsp;Mary R. Starich,&nbsp;Nico Tjandra,&nbsp;Pushpa Mishra","doi":"10.1007/s10858-021-00359-9","DOIUrl":"https://doi.org/10.1007/s10858-021-00359-9","url":null,"abstract":"<p>Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand–protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous ¹H-¹⁵?N, ¹H-¹³C SESAME based pulse scheme for the rapid acquisition of ¹H^C/N-R₂ relaxation rates for the determination of backbone and sidechain PREs of proteins. The ¹H^N-R₂ rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R²?=?0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00359-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4928532","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":"Protein in-cell NMR spectroscopy at 1.2 GHz","authors":"Enrico Luchinat,&nbsp;Letizia Barbieri,&nbsp;Matteo Cremonini,&nbsp;Lucia Banci","doi":"10.1007/s10858-021-00358-w","DOIUrl":"https://doi.org/10.1007/s10858-021-00358-w","url":null,"abstract":"<p>In-cell NMR spectroscopy provides precious structural and functional information on biological macromolecules in their native cellular environment at atomic resolution. However, the intrinsic low sensitivity of NMR imposes a big limitation in the applicability of the methodology. In this respect, the recently developed commercial 1.2?GHz NMR spectrometer is expected to introduce significant benefits. However, cell samples may suffer from detrimental effects at ultrahigh fields, that must be carefully evaluated. Here we show the first in-cell NMR spectra recorded at 1.2?GHz on human cells, and we compare resolution and sensitivity against those obtained at 900 and 950?MHz. To evaluate the effects of different spin relaxation rates, SOFAST-HMQC and BEST-TROSY spectra were recorded on intracellular α-synuclein and carbonic anhydrase. Major improvements are observed at 1.2?GHz when analyzing unfolded proteins, such as α-synuclein, while the TROSY scheme improves the resolution for both globular and unfolded proteins.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00358-w","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4493247","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 simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks","authors":"Mengli Cai,&nbsp;Ying Huang,&nbsp;John Lloyd,&nbsp;Robert Craigie,&nbsp;G. Marius Clore","doi":"10.1007/s10858-021-00357-x","DOIUrl":"https://doi.org/10.1007/s10858-021-00357-x","url":null,"abstract":"<p>A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val ¹H/¹³C methyl protonated proteins from 100?ml cultures in M9?++?/D₂O medium induced at high (OD₆₀₀?~?10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of ²H/¹⁵N/¹³C and ¹H/¹³C labeled proteins, yields comparable quantities of protein from 100?ml cell culture to those obtained using a conventional 1 L culture with M9/D₂O medium, while using three-fold less α-ketoisovaleric (1,2,3,4-¹³C₄; 3,4′,4′,4′-d₄) and α-ketobutyric (¹³C₄; 3,3-d₂) acid precursors.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-021-00357-x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4159519","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 production of a functional G protein-coupled receptor in E. coli for structural studies","authors":"Layara Akemi Abiko,&nbsp;Marco Rogowski,&nbsp;Antoine Gautier,&nbsp;Gebhard Schertler,&nbsp;Stephan Grzesiek","doi":"10.1007/s10858-020-00354-6","DOIUrl":"https://doi.org/10.1007/s10858-020-00354-6","url":null,"abstract":"<p>G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in <i>E. coli</i> has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR <i>E. coli</i> expression and then describe the development of an optimized robust protocol for the <i>E. coli</i> expression and purification of two mutants of the turkey β₁-adrenergic receptor (β₁AR) uniformly or selectively labeled in ¹⁵N or ²H,¹⁵N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for <i>E. coli</i> expression. Optimization of <i>E. coli</i> expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2–0.3?mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-β₁AR mutant also comprises the two native tyrosines Y^5.58 and Y^7.53, which enable G protein binding. High-quality ¹H-¹⁵N TROSY spectra were obtained for <i>E. coli</i>-expressed YY-β₁AR in three different functional states (antagonist, agonist, and agonist?+?G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-020-00354-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5044170","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":"On the use of 3J-coupling NMR data to derive structural information on proteins","authors":"Lorna J. Smith,&nbsp;Wilfred F. van Gunsteren,&nbsp;Bartosz Stankiewicz,&nbsp;Niels Hansen","doi":"10.1007/s10858-020-00355-5","DOIUrl":"https://doi.org/10.1007/s10858-020-00355-5","url":null,"abstract":"<p>Values of ³<i>J</i>-couplings as obtained from NMR experiments on proteins cannot easily be used to determine protein structure due to the difficulty of accounting for the high sensitivity of intermediate ³<i>J</i>-coupling values (4–8?Hz) to the averaging period that must cover the conformational variability of the torsional angle related to the ³<i>J</i>-coupling, and due to the difficulty of handling the multiple-valued character of the inverse Karplus relation between torsional angle and ³<i>J</i>-coupling. Both problems can be solved by using ³<i>J</i>-coupling time-averaging local-elevation restraining MD simulation. Application to the protein hen egg white lysozyme using 213 backbone and side-chain ³<i>J</i>-coupling restraints shows that a conformational ensemble compatible with the experimental data can be obtained using this technique, and that accounting for averaging and the ability of the algorithm to escape from local minima for the torsional angle induced by the Karplus relation, are essential for a comprehensive use of ³<i>J</i>-coupling data in protein structure determination.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-020-00355-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5338680","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":"Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy","authors":"Arthur Hinterholzer,&nbsp;Vesna Stanojlovic,&nbsp;Christof Regl,&nbsp;Christian G. Huber,&nbsp;Chiara Cabrele,&nbsp;Mario Schubert","doi":"10.1007/s10858-020-00356-4","DOIUrl":"https://doi.org/10.1007/s10858-020-00356-4","url":null,"abstract":"<p>The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of?+?18?Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D?NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pK_a values of isoAsp and C-terminal Asp in short peptides. The characteristic ¹H-¹³C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4–5) and 40?°C. The results show that the application of our 2D?NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-020-00356-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4823097","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":"Correction to: A suite of 19F based relaxation dispersion experiments to assess biomolecular motions","authors":"Jan H. Overbeck,&nbsp;Werner Kremer,&nbsp;Remco Sprangers","doi":"10.1007/s10858-020-00352-8","DOIUrl":"https://doi.org/10.1007/s10858-020-00352-8","url":null,"abstract":"","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-020-00352-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5370578","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":"Correction to: NMR in pharmaceutical discovery and development","authors":"Raymond S. Norton,&nbsp;Wolfgang Jahnke","doi":"10.1007/s10858-020-00351-9","DOIUrl":"https://doi.org/10.1007/s10858-020-00351-9","url":null,"abstract":"","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-020-00351-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4551653","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}