Pub Date : 2024-06-01Epub Date: 2024-02-29DOI: 10.1007/s10858-024-00437-8
Bharat P Chaudhary, Jochem Struppe, Hem Moktan, David Zoetewey, Donghua H Zhou, Smita Mohanty
N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-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.
{"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, Jochem Struppe, Hem Moktan, David Zoetewey, Donghua H Zhou, Smita Mohanty","doi":"10.1007/s10858-024-00437-8","DOIUrl":"10.1007/s10858-024-00437-8","url":null,"abstract":"<p><p>N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-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>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-06-01","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}
Pub Date : 2024-05-24DOI: 10.1007/s10858-024-00441-y
Alexandra Locke, Kylee Guarino, Gordon S Rule
A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and 2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain 1H-13C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100-150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.
{"title":"Labeling of methyl groups: a streamlined protocol and guidance for the selection of <sup>2</sup>H precursors based on molecular weight.","authors":"Alexandra Locke, Kylee Guarino, Gordon S Rule","doi":"10.1007/s10858-024-00441-y","DOIUrl":"10.1007/s10858-024-00441-y","url":null,"abstract":"<p><p>A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D<sub>2</sub>O and <sup>2</sup>H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D<sub>2</sub>O) or deuterated glucose is not required to obtain <sup>1</sup>H-<sup>13</sup>C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100-150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141086372","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}
Pub Date : 2024-03-30DOI: 10.1007/s10858-024-00439-6
Benxun Pan, Canyong Guo, Dongsheng Liu, Kurt Wüthrich
In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the "toggle switch tryptophan". Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.
{"title":"Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression.","authors":"Benxun Pan, Canyong Guo, Dongsheng Liu, Kurt Wüthrich","doi":"10.1007/s10858-024-00439-6","DOIUrl":"https://doi.org/10.1007/s10858-024-00439-6","url":null,"abstract":"<p><p>In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, <sup>19</sup>F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The <sup>19</sup>F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the \"toggle switch tryptophan\". Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140329463","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}
Pub Date : 2024-03-28DOI: 10.1007/s10858-023-00433-4
Arthur Giraud, Lionel Imbert, Adrien Favier, Faustine Henot, Francis Duffieux, Camille Samson, Oriane Frances, Elodie Crublet, Jérôme Boisbouvier
Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [15N, 13C, 2H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1H2O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1HN, 15N, 13C′, 13Cα, 13Cβ resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs’ higher order structures, dynamics and interactions, using solution-state NMR.
单克隆抗体(mAbs)是一种生物治疗药物,在治疗许多危及生命的疾病和慢性疾病方面取得了巨大成功。抗原的识别由片段抗原结合(Fab)区域介导,该区域由四个不同的二硫桥连接的免疫球蛋白结构域组成。核磁共振是评估 Fabs 的完整性、结构和相互作用的一种强有力的方法,但迄今为止,由于 Fabs 的大小和缺乏生产同位素标记样品的方法,特定位点分析一直受到阻碍。我们在此提出了一种高效的体外方法来制备[15N, 13C, 2H]标记的 Fabs,从而能够对这些强大的治疗药物进行高分辨率核磁共振研究。作为一种开放式系统,无细胞表达模式可在二硫键异构酶存在的情况下对氧化还原电位进行微调控制,以增强原生二硫键的形成。此外,抑制 S30 无细胞提取物中的转氨酶,就有机会在 1H2O 培养基中直接制备过氘化的 Fab 样品,而无需耗时且低效的重折叠过程。我们采用这种特定的方法制备出了最佳标记的治疗用 Fab 样品,从而能够对全长 Fab 的 1HN、15N、13C′、13Cα、13Cβ 共振进行顺序赋值。成功分配了针对人类 LAMP1 糖蛋白的 Fab 结构域 90% 的骨架共振,为利用溶液态核磁共振以原子分辨率研究 Fab 的高阶结构、动力学和相互作用提供了新的机会。
{"title":"Enabling site-specific NMR investigations of therapeutic Fab using a cell-free based isotopic labeling approach: application to anti-LAMP1 Fab","authors":"Arthur Giraud, Lionel Imbert, Adrien Favier, Faustine Henot, Francis Duffieux, Camille Samson, Oriane Frances, Elodie Crublet, Jérôme Boisbouvier","doi":"10.1007/s10858-023-00433-4","DOIUrl":"https://doi.org/10.1007/s10858-023-00433-4","url":null,"abstract":"<p>Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [<sup>15</sup>N, <sup>13</sup>C, <sup>2</sup>H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in <sup>1</sup>H<sub>2</sub>O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of <sup>1</sup>H<sub>N</sub>, <sup>15</sup>N, <sup>13</sup>C′, <sup>13</sup>C<sub>α</sub>, <sup>13</sup>C<sub>β</sub> resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs’ higher order structures, dynamics and interactions, using solution-state NMR.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140315702","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}
Pub Date : 2024-03-21DOI: 10.1007/s10858-024-00440-z
Giorgia Toscano, Julian Holzinger, Benjamin Nagl, Georg Kontaxis, Hanspeter Kählig, Robert Konrat, Roman J Lichtenecker
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 Escherichia coli overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.
{"title":"Decorating phenylalanine side-chains with triple labeled <sup>13</sup>C/<sup>19</sup>F/<sup>2</sup>H isotope patterns.","authors":"Giorgia Toscano, Julian Holzinger, Benjamin Nagl, Georg Kontaxis, Hanspeter Kählig, Robert Konrat, Roman J Lichtenecker","doi":"10.1007/s10858-024-00440-z","DOIUrl":"https://doi.org/10.1007/s10858-024-00440-z","url":null,"abstract":"<p><p>We present an economic and straightforward method to introduce <sup>13</sup>C-<sup>19</sup>F 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-<sup>13</sup>C, 2,3,5,6-<sup>2</sup>H<sub>4</sub>] 4-fluorophenylalanine from the commercially available isotope sources [2-<sup>13</sup>C] acetone and deuterium oxide. This compound is readily metabolized by standard Escherichia coli overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-01Epub Date: 2023-11-21DOI: 10.1007/s10858-023-00428-1
Alexander R Davis, Elijah T Roberts, I Jonathan Amster, Adam W Barb
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 E. coli culture. We developed a system utilizing Saccharomyces cerevisiae, [13C]-glucose, and [15N]-ammonium chloride with complexity comparable to E. coli. 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 E. coli.
{"title":"Uniform [<sup>13</sup>C,<sup>15</sup>N]-labeled and glycosylated IgG1 Fc expressed in Saccharomyces cerevisiae.","authors":"Alexander R Davis, Elijah T Roberts, I Jonathan Amster, Adam W Barb","doi":"10.1007/s10858-023-00428-1","DOIUrl":"10.1007/s10858-023-00428-1","url":null,"abstract":"<p><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 E. coli culture. We developed a system utilizing Saccharomyces cerevisiae, [<sup>13</sup>C]-glucose, and [<sup>15</sup>N]-ammonium chloride with complexity comparable to E. coli. 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% <sup>15</sup>N and 30% <sup>13</sup>C incorporation into the polypeptide, or a rich medium which provides 98% <sup>15</sup>N and 95% <sup>13</sup>C 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 E. coli.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11025670/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-01Epub Date: 2024-01-03DOI: 10.1007/s10858-023-00431-6
Ved Prakash Tiwari, Debajyoti De, Nemika Thapliyal, Lewis E Kay, Pramodh Vallurupalli
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 ( ~ 550 s-1, ~ 1200 s-1, ~ 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.
尽管核磁共振光谱被常规用于研究生物大分子的构象动态,但在交换比双态更复杂的情况下,例如当一个 "可见 "的主要构象与两个 "不可见 "的次要态在毫秒级的时间尺度上发生交换时,对数据的可靠分析就面临挑战。化学交换饱和转移(CEST)核磁共振实验最初是为了研究发生缓慢相互转换的系统而开发的,但它对中快时间尺度的生物分子构象交换也很敏感,这一点正变得越来越清楚。在这里,我们通过研究人 HYPA/FBP11 的 FF 结构域(WT FF)的折叠 (F) 状态与其两个折叠中间体 I1 和 I2 之间的相互转换,探讨了酰胺 15N CEST 实验在表征毫秒级蛋白质三态交换方面的用途。尽管 15N CPMG 实验与 F 状态在毫秒时间尺度上与单个次要状态相互转化一致,但 15N CEST 数据清楚地确定了 F 与一对次要状态之间的交换过程。对在单一温度下记录的 15N CEST 数据进行分析,无法得到一个独特的三态交换模型。然而,在对多个温度下记录的 CEST 数据进行简单的两态分析的基础上,将两个次态的化学位移差异的相对符号包括在内,就可以得到一个稳健的三态模型,其中 F、I1 和 I2 态在毫秒级([公式:见正文] ~ 550 s-1,[公式:见正文] ~ 1200 s-1,[公式:见正文] ~ 5000 s-1)上相互转换,I1 和 I2 态的稀疏程度分别为 ~ 0.在 15 °C 时,I1 和 I2 的稀疏度分别为 ~ 0.15% 和 ~ 0.35%。从 CEST 数据中提取最佳拟合交换参数不需要对交换参数空间进行计算要求极高的网格搜索。因此,CEST 实验的实用性远远超出了对核磁共振化学位移时间尺度上缓慢交换的构象的研究,还包括了相互转换速率达到数千/秒数量级的系统。
{"title":"Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale.","authors":"Ved Prakash Tiwari, Debajyoti De, Nemika Thapliyal, Lewis E Kay, Pramodh Vallurupalli","doi":"10.1007/s10858-023-00431-6","DOIUrl":"10.1007/s10858-023-00431-6","url":null,"abstract":"<p><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 <sup>15</sup>N 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 <sup>15</sup>N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, <sup>15</sup>N 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 <sup>15</sup>N 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 ( <math><msub><mi>k</mi> <mrow><mi>e</mi> <mi>x</mi> <mo>,</mo> <mi>F</mi> <mi>I</mi> <mn>1</mn></mrow> </msub> </math> ~ 550 s<sup>-1</sup>, <math><msub><mi>k</mi> <mrow><mi>e</mi> <mi>x</mi> <mo>,</mo> <mi>F</mi> <mi>I</mi> <mn>2</mn></mrow> </msub> </math> ~ 1200 s<sup>-1</sup>, <math><msub><mi>k</mi> <mrow><mi>e</mi> <mi>x</mi> <mo>,</mo> <mi>I</mi> <mn>1</mn> <mi>I</mi> <mn>2</mn></mrow> </msub> </math> ~ 5000 s<sup>-1</sup>), 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>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-03-01","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}
Pub Date : 2024-03-01Epub Date: 2023-10-11DOI: 10.1007/s10858-023-00427-2
Theresa Höfurthner, Giorgia Toscano, Georg Kontaxis, Andreas Beier, Moriz Mayer, Leonhard Geist, Darryl B McConnell, Harald Weinstabl, Roman Lichtenecker, Robert Konrat
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.
{"title":"Synthesis of a <sup>13</sup>C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics.","authors":"Theresa Höfurthner, Giorgia Toscano, Georg Kontaxis, Andreas Beier, Moriz Mayer, Leonhard Geist, Darryl B McConnell, Harald Weinstabl, Roman Lichtenecker, Robert Konrat","doi":"10.1007/s10858-023-00427-2","DOIUrl":"10.1007/s10858-023-00427-2","url":null,"abstract":"<p><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 <sup>13</sup>C-<sup>1</sup> H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased <sup>13</sup>C-T<sub>2</sub> 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>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-03-01","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}
Pub Date : 2024-03-01Epub Date: 2023-12-20DOI: 10.1007/s10858-023-00432-5
Pauline Defant, Christof Regl, Christian G Huber, Mario Schubert
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.
{"title":"The NMR signature of maltose-based glycation in full-length proteins.","authors":"Pauline Defant, Christof Regl, Christian G Huber, Mario Schubert","doi":"10.1007/s10858-023-00432-5","DOIUrl":"10.1007/s10858-023-00432-5","url":null,"abstract":"<p><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-d<sub>4</sub> in D<sub>2</sub>O. 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>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-03-01","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}
Pub Date : 2024-02-26DOI: 10.1007/s10858-024-00438-7
Kieran T. Cockburn, Brian D. Sykes
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.
{"title":"Fluorine labelling for in situ 19F NMR in oriented systems","authors":"Kieran T. Cockburn, Brian D. Sykes","doi":"10.1007/s10858-024-00438-7","DOIUrl":"https://doi.org/10.1007/s10858-024-00438-7","url":null,"abstract":"<p>The focus of this project is to take advantage of the large NMR chemical shift anisotropy of <sup>19</sup>F 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 CF<sub>3</sub> 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 <sup>19</sup>F NMR spectrum. The potential applications are widespread and not limited to biological applications.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"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}