Journal of Biomolecular NMR最新文献

{"title":"Decorating phenylalanine side-chains with triple labeled 13C/19F/2H isotope patterns","authors":"Giorgia Toscano,&nbsp;Julian Holzinger,&nbsp;Benjamin Nagl,&nbsp;Georg Kontaxis,&nbsp;Hanspeter Kählig,&nbsp;Robert Konrat,&nbsp;Roman J. Lichtenecker","doi":"10.1007/s10858-024-00440-z","DOIUrl":"10.1007/s10858-024-00440-z","url":null,"abstract":"<div><p>We present an economic and straightforward method to introduce ¹³C-¹⁹F spin systems into the deuterated aromatic side chains of phenylalanine as reporters for various protein NMR applications. The method is based on the synthesis of [4-¹³C, 2,3,5,6-²H₄] 4-fluorophenylalanine from the commercially available isotope sources [2-¹³C] acetone and deuterium oxide. This compound is readily metabolized by standard <i>Escherichia coli</i> overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 3","pages":"139 - 147"},"PeriodicalIF":1.3,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-024-00440-z.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140178941","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":"Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR","authors":"Bharat P. Chaudhary,&nbsp;Jochem Struppe,&nbsp;Hem Moktan,&nbsp;David Zoetewey,&nbsp;Donghua H. Zhou,&nbsp;Smita Mohanty","doi":"10.1007/s10858-024-00437-8","DOIUrl":"10.1007/s10858-024-00437-8","url":null,"abstract":"<div><p><i>N</i>-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in <i>N</i>-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein. Oligosaccharyltransferase (OST), a multi-subunit membrane embedded enzyme catalyzes this glycosylation reaction in eukaryotes. In yeast, Ost4 is the smallest of nine subunits and bridges the interaction of the catalytic subunit, Stt3, with Ost3 (or its homolog, Ost6). Mutations of any C-terminal hydrophobic residues in Ost4 to a charged residue destabilizes the enzyme and negatively impacts its function. Specifically, the V23D mutation results in a temperature-sensitive phenotype in yeast. Here, we report the reconstitution of both purified recombinant Ost4 and Ost4V23D each in a POPC/POPE lipid bilayer and their resonance assignments using heteronuclear 2D and 3D solid-state NMR with magic-angle spinning. The chemical shifts of Ost4 changed significantly upon the V23D mutation, suggesting a dramatic change in its chemical environment.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 2","pages":"109 - 117"},"PeriodicalIF":1.3,"publicationDate":"2024-02-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139988971","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":"Fluorine labelling for in situ 19F NMR in oriented systems","authors":"Kieran T. Cockburn,&nbsp;Brian D. Sykes","doi":"10.1007/s10858-024-00438-7","DOIUrl":"10.1007/s10858-024-00438-7","url":null,"abstract":"<div><p>The focus of this project is to take advantage of the large NMR chemical shift anisotropy of ¹⁹F to determine the orientation of fluorine labeled biomolecules in situ in oriented biological systems such as muscle. The difficulty with a single fluorine atom is that the orientation determined from a chemical shift is not singlevalued in the case of a fully anisotropic chemical shift tensor. The utility of a labeling approach with two fluorine labels in a fixed molecular framework where one of the labels has an axially symmetric chemical shift anisotropy such as a CF₃ group and the other has a fully asymmetric chemical shift anisotropy such as 5-fluorotryptophan is evaluated. The result is that the orientation of the label can be determined straightforwardly from a single one-dimensional ¹⁹F NMR spectrum. The potential applications are widespread and not limited to biological applications.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 2","pages":"119 - 124"},"PeriodicalIF":1.3,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139968236","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 comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy","authors":"Ganesh P. Subedi,&nbsp;Elijah T. Roberts,&nbsp;Alexander R. Davis,&nbsp;Paul G. Kremer,&nbsp;I. Jonathan Amster,&nbsp;Adam W. Barb","doi":"10.1007/s10858-023-00434-3","DOIUrl":"10.1007/s10858-023-00434-3","url":null,"abstract":"<div><p>A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[¹⁵N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for ¹⁵N-labeling in HEK293 cells. </p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 2","pages":"125 - 132"},"PeriodicalIF":1.3,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139968748","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":"Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale","authors":"Ved Prakash Tiwari,&nbsp;Debajyoti De,&nbsp;Nemika Thapliyal,&nbsp;Lewis E. Kay,&nbsp;Pramodh Vallurupalli","doi":"10.1007/s10858-023-00431-6","DOIUrl":"10.1007/s10858-023-00431-6","url":null,"abstract":"<div><p>Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a ‘visible’ major conformer exchanges with two ‘invisible’ minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate—fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide ¹⁵N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although ¹⁵N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, ¹⁵N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of ¹⁵N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale (<span>({k}_{ex,FI1})</span> ~ 550 s⁻¹, <span>({k}_{ex,FI2})</span> ~ 1200 s⁻¹, <span>({k}_{ex,I1I2})</span> ~ 5000 s⁻¹), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15 °C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 1","pages":"39 - 60"},"PeriodicalIF":1.3,"publicationDate":"2024-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139085430","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":"The NMR signature of maltose-based glycation in full-length proteins","authors":"Pauline Defant,&nbsp;Christof Regl,&nbsp;Christian G. Huber,&nbsp;Mario Schubert","doi":"10.1007/s10858-023-00432-5","DOIUrl":"10.1007/s10858-023-00432-5","url":null,"abstract":"<div><p>Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose. For better comparison, data collection was done under denaturing conditions using 7 M urea-d₄ in D₂O. The here presented correlation patterns serve as a signature and can be used to identify maltose-based glycation in any protein that can be denatured. In addition to the model protein BSA, which can be readily glycated, we present data of the biotherapeutic abatacept containing maltose in its formulation buffer. With this contribution, we demonstrate that NMR spectroscopy is an independent method for detecting maltose-based glycation, that is suited for cross-validation with other methods.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 1","pages":"61 - 72"},"PeriodicalIF":1.3,"publicationDate":"2023-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10981599/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138796146","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":"Short and long range 2D 15N–15N NMR correlations among peptide groups by novel solid state dipolar mixing schemes","authors":"Sungsool Wi,&nbsp;Conggang Li,&nbsp;Karen Pham,&nbsp;Woonghee Lee,&nbsp;Lucio Frydman","doi":"10.1007/s10858-023-00429-0","DOIUrl":"10.1007/s10858-023-00429-0","url":null,"abstract":"<div><p>A recently developed homonuclear dipolar recoupling scheme, Adiabatic Linearly FREquency Swept reCOupling (AL FRESCO), was applied to record two-dimensional (2D) ¹⁵N–¹⁵N correlations on uniformly ¹⁵N-labeled GB1 powders. A major feature exploited in these ¹⁵N–¹⁵N correlations was AL FRESCO’s remarkably low RF power demands, which enabled seconds-long mixing schemes when establishing direct correlations. These ¹⁵N–¹⁵N mixing schemes proved efficient regardless of the magic-angle spinning (MAS) rate and, being nearly free from dipolar truncation effects, they enabled the detection of long-range, weak dipolar couplings, even in the presence of strong short-range dipolar couplings. This led to a connectivity information that was significantly better than that obtained with spontaneously proton-driven, ¹⁵N spin-diffusion experiments. An indirect approach producing long-range ¹⁵N–¹⁵N correlations was also tested, relying on short (ms-long) ¹H^N–¹H^N mixings schemes while applying AL FRESCO chirped pulses along the ¹⁵N channel. These indirect mixing schemes produced numerous long-distance N_i–N_i±n (n = 2 − 5) correlations, that might be useful for characterizing three-dimensional arrangements in proteins. Once again, these AL FRESCO mediated experiments proved more informative than variants based on spin-diffusion-based ¹H^N–¹H^N counterparts.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 1","pages":"19 - 30"},"PeriodicalIF":1.3,"publicationDate":"2023-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138691562","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":"19F-NMR studies of the impact of different detergents and nanodiscs on the A2A adenosine receptor","authors":"Francisco Mendoza-Hoffmann,&nbsp;Canyong Guo,&nbsp;Yanzhuo Song,&nbsp;Dandan Feng,&nbsp;Lingyun Yang,&nbsp;Kurt Wüthrich","doi":"10.1007/s10858-023-00430-7","DOIUrl":"10.1007/s10858-023-00430-7","url":null,"abstract":"<div><p>For the A_2A adenosine receptor (A_2AAR), a class A G-protein-coupled receptor (GPCR), reconstituted in <i>n</i>-dodecyl-<i>β</i>-D-maltoside (DDM)/‌‌‌‌‌cholesteryl hemisuccinate (CHS) mixed micelles, previous ¹⁹F-NMR studies revealed the presence of multiple simultaneously populated conformational states. Here, we study the influence of a different detergent, lauryl maltose neopentyl glycol (LMNG) in mixed micelles with CHS, and of lipid bilayer nanodiscs on these conformational equilibria. The populations of locally different substates are pronouncedly different in DDM/‌‌‌‌‌CHS and LMNG/‌‌‌‌‌CHS micelles, whereas the A_2AAR conformational manifold in LMNG/‌‌‌‌‌CHS micelles is closely similar to that in the lipid bilayer nanodiscs. Considering that nanodiscs represent a closer match of the natural lipid bilayer membrane, these observations support that LMNG/‌‌‌‌‌CHS micelles are a good choice for reconstitution trials of class A GPCRs for NMR studies in solution.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 1","pages":"31 - 37"},"PeriodicalIF":1.3,"publicationDate":"2023-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138585223","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":"Uniform [13C,15N]-labeled and glycosylated IgG1 Fc expressed in Saccharomyces cerevisiae","authors":"Alexander R. Davis,&nbsp;Elijah T. Roberts,&nbsp;I. Jonathan Amster,&nbsp;Adam W. Barb","doi":"10.1007/s10858-023-00428-1","DOIUrl":"10.1007/s10858-023-00428-1","url":null,"abstract":"<div><p>Despite the prevalence and importance of glycoproteins in human biology, methods for isotope labeling suffer significant limitations. Common prokaryotic platforms do not produce mammalian post-translation modifications that are essential to the function of many human glycoproteins, including immunoglobulin G1 (IgG1). Mammalian expression systems require complex media and thus introduce significant costs to achieve uniform labeling. Expression with Pichia is available, though expertise and equipment requirements surpass <i>E. coli</i> culture. We developed a system utilizing <i>Saccharomyces cerevisiae</i>, [¹³C]-glucose, and [¹⁵N]-ammonium chloride with complexity comparable to <i>E. coli</i>. Here we report two vectors for expressing the crystallizable fragment (Fc) of IgG1 for secretion into the culture medium, utilizing the ADH2 or DDI2 promoters. We also report a strategy to optimize the expression yield using orthogonal Taguchi arrays. Lastly, we developed two different media formulations, a standard medium which provides 86–92% ¹⁵N and 30% ¹³C incorporation into the polypeptide, or a rich medium which provides 98% ¹⁵N and 95% ¹³C incorporation as determined by mass spectrometry. This advance represents an expression and optimization strategy accessible to experimenters with the capability to grow and produce proteins for NMR-based experiments using <i>E. coli</i>.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 1","pages":"9 - 18"},"PeriodicalIF":1.3,"publicationDate":"2023-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138290066","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":"Efficient determination of the accessible conformation space of multi-domain complexes based on EPR PELDOR data","authors":"Sina Kazemi,&nbsp;Anna Lopata,&nbsp;Andreas Kniss,&nbsp;Lukas Pluska,&nbsp;Peter Güntert,&nbsp;Thomas Sommer,&nbsp;Thomas F. Prisner,&nbsp;Alberto Collauto,&nbsp;Volker Dötsch","doi":"10.1007/s10858-023-00426-3","DOIUrl":"10.1007/s10858-023-00426-3","url":null,"abstract":"<div><p>Many proteins can adopt multiple conformations which are important for their function. This is also true for proteins and domains that are covalently linked to each other. One important example is ubiquitin, which can form chains of different conformations depending on which of its lysine side chains is used to form an isopeptide bond with the C-terminus of another ubiquitin molecule. Similarly, ubiquitin gets covalently attached to active-site residues of E2 ubiquitin-conjugating enzymes. Due to weak interactions between ubiquitin and its interaction partners, these covalent complexes adopt multiple conformations. Understanding the function of these complexes requires the characterization of the entire accessible conformation space and its modulation by interaction partners. Long-range (1.8–10 nm) distance restraints obtained by EPR spectroscopy in the form of probability distributions are ideally suited for this task as not only the mean distance but also information about the conformation dynamics is encoded in the experimental data. Here we describe a computational method that we have developed based on well-established structure determination software using NMR restraints to calculate the accessible conformation space using PELDOR/DEER data.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"77 5-6","pages":"261 - 269"},"PeriodicalIF":2.7,"publicationDate":"2023-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10687113/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"107590024","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}