Pub Date : 2022-01-08DOI: 10.1007/s10858-021-00388-4
Ümit Akbey
Determination of protein structure and dynamics is key to understand the mechanism of protein action. Perdeuterated proteins have been used to obtain high resolution/sensitivty NMR experiments via proton-detection. These methods utilizes 1H, 13C and 15N nuclei for chemical shift dispersion or relaxation probes, despite the existing abundant deuterons. However, a high-sensitivity NMR method to utilize deuterons and e.g. determine site-specific deuterium quadrupolar pattern information has been lacking due to technical difficulties associated with deuterium’s large quadrupolar couplings. Here, we present a novel deuterium-excited and proton-detected three-dimensional 2H–13C–1H MAS NMR experiment to utilize deuterons and to obtain site-specific methyl 2H quadrupolar patterns on detuterated proteins for the first time. A high-resolution fingerprint 1H–15N HSQC-spectrum is correlated with the anisotropic deuterium quadrupolar tensor in the third dimension. Results from a model perdeuterated protein has been shown.
确定蛋白质的结构和动力学是了解蛋白质作用机制的关键。氘化蛋白已被用于通过质子检测获得高分辨率/灵敏度的核磁共振实验。这些方法利用1H, 13C和15N核进行化学位移色散或弛豫探针,尽管存在丰富的氘核。然而,由于与氘的大四极耦合相关的技术困难,缺乏一种高灵敏度的核磁共振方法来利用氘,例如确定特定位点的氘四极模式信息。在这里,我们提出了一种新的氘激发和质子检测的三维2H - 13c - 1h MAS NMR实验,首次利用氘核并获得了氘化蛋白上特定位点的甲基2H四极性模式。高分辨率指纹图谱1H-15N hsqc谱在三维空间上与各向异性氘四极张量相关。从一个模型的结果已被显示。
{"title":"Site-specific protein methyl deuterium quadrupolar patterns by proton-detected 3D 2H–13C–1H MAS NMR spectroscopy","authors":"Ümit Akbey","doi":"10.1007/s10858-021-00388-4","DOIUrl":"10.1007/s10858-021-00388-4","url":null,"abstract":"<div><p>Determination of protein structure and dynamics is key to understand the mechanism of protein action. Perdeuterated proteins have been used to obtain high resolution/sensitivty NMR experiments via proton-detection. These methods utilizes <sup>1</sup>H, <sup>13</sup>C and <sup>15</sup>N nuclei for chemical shift dispersion or relaxation probes, despite the existing abundant deuterons. However, a high-sensitivity NMR method to utilize deuterons and e.g. determine site-specific deuterium quadrupolar pattern information has been lacking due to technical difficulties associated with deuterium’s large quadrupolar couplings. Here, we present a novel deuterium-excited and proton-detected three-dimensional <sup>2</sup>H–<sup>13</sup>C–<sup>1</sup>H MAS NMR experiment to utilize deuterons and to obtain site-specific methyl <sup>2</sup>H quadrupolar patterns on detuterated proteins for the first time. A high-resolution fingerprint <sup>1</sup>H–<sup>15</sup>N HSQC-spectrum is correlated with the anisotropic deuterium quadrupolar tensor in the third dimension. Results from a model perdeuterated protein has been shown.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2022-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4655941","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}
NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.
{"title":"Monitoring protein unfolding transitions by NMR-spectroscopy","authors":"Matthias Dreydoppel, Jochen Balbach, Ulrich Weininger","doi":"10.1007/s10858-021-00389-3","DOIUrl":"10.1007/s10858-021-00389-3","url":null,"abstract":"<div><p>NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2022-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00389-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4502881","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}
Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with uniformly isotope-labeled glycoproteins due to the large quantity of labeled l-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1.
{"title":"Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1","authors":"Saeko Yanaka, Hirokazu Yagi, Rina Yogo, Masayoshi Onitsuka, Koichi Kato","doi":"10.1007/s10858-021-00387-5","DOIUrl":"10.1007/s10858-021-00387-5","url":null,"abstract":"<div><p>Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with uniformly isotope-labeled glycoproteins due to the large quantity of labeled <span>l</span>-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2022-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4121049","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 : 2021-11-23DOI: 10.1007/s10858-021-00386-6
Alons Lends, Mélanie Berbon, Birgit Habenstein, Yusuke Nishiyama, Antoine Loquet
Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of proton (1H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of 1H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) 13C spectroscopy, detected on 1H, is proposed for fast MAS regime (> 60 kHz) to perform the sequential assignment of insoluble proteins of small size, without any specific deuteration requirement. By combining two three-dimensional 1H detected experiments correlating a 13C DQ dimension respectively to its intra-residue and sequential 15 N-1H pairs, a sequential walk through DQ (Ca + CO) resonance is obtained. The approach takes advantage of fast MAS to achieve an efficient sensitivity and the addition of a DQ dimension provides spectral features useful for the resonance assignment process.
固体核磁共振波谱是一种强大的技术,可以在原子分辨率上研究不溶性和非晶体蛋白质和蛋白质复合物。快速魔角旋转(MAS)质子(1H)检测技术的发展大大提高了该技术的分析能力,使在几小时内获得1H检测指纹实验成为可能。本文提出了一种基于双量子(DQ) 13C光谱的方法,在1H上检测,用于快速MAS状态(> 60 kHz),以执行小尺寸不溶性蛋白质的顺序分配,而不需要任何特定的氘化要求。通过结合两个三维1H检测实验,分别将13C DQ维度与其残基内和顺序的15对N-1H对相关联,获得了DQ (Ca + CO)共振的顺序遍历。该方法利用快速MAS实现了高效的灵敏度,并且增加了DQ维,为共振分配过程提供了有用的光谱特征。
{"title":"Protein resonance assignment by solid-state NMR based on 1H-detected 13C double-quantum spectroscopy at fast MAS","authors":"Alons Lends, Mélanie Berbon, Birgit Habenstein, Yusuke Nishiyama, Antoine Loquet","doi":"10.1007/s10858-021-00386-6","DOIUrl":"10.1007/s10858-021-00386-6","url":null,"abstract":"<div><p>Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of proton (<sup>1</sup>H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of <sup>1</sup>H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) <sup>13</sup>C spectroscopy, detected on <sup>1</sup>H, is proposed for fast MAS regime (> 60 kHz) to perform the sequential assignment of insoluble proteins of small size, without any specific deuteration requirement. By combining two three-dimensional <sup>1</sup>H detected experiments correlating a <sup>13</sup>C DQ dimension respectively to its intra-residue and sequential <sup>15</sup> N-<sup>1</sup>H pairs, a sequential walk through DQ (Ca + CO) resonance is obtained. The approach takes advantage of fast MAS to achieve an efficient sensitivity and the addition of a DQ dimension provides spectral features useful for the resonance assignment process.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00386-6.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4909078","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 : 2021-11-05DOI: 10.1007/s10858-021-00385-7
Paweł Kasprzak, Mateusz Urbańczyk, Krzysztof Kazimierczuk
Non-uniform sampling (NUS) is a popular way of reducing the amount of time taken by multidimensional NMR experiments. Among the various non-uniform sampling schemes that exist, the Poisson-gap (PG) schedules are particularly popular, especially when combined with compressed-sensing (CS) reconstruction of missing data points. However, the use of PG is based mainly on practical experience and has not, as yet, been explained in terms of CS theory. Moreover, an apparent contradiction exists between the reported effectiveness of PG and CS theory, which states that a “flat” pseudo-random generator is the best way to generate sampling schedules in order to reconstruct sparse spectra. In this paper we explain how, and in what situations, PG reveals its superior features in NMR spectroscopy. We support our theoretical considerations with simulations and analyses of experimental data from the Biological Magnetic Resonance Bank (BMRB). Our analyses reveal a previously unnoticed feature of many NMR spectra that explains the success of ”blue-noise” schedules, such as PG. We call this feature “clustered sparsity”. This refers to the fact that the peaks in NMR spectra are not just sparse but often form clusters in the indirect dimension, and PG is particularly suited to deal with such situations. Additionally, we discuss why denser sampling in the initial and final parts of the clustered signal may be useful.
{"title":"Clustered sparsity and Poisson-gap sampling","authors":"Paweł Kasprzak, Mateusz Urbańczyk, Krzysztof Kazimierczuk","doi":"10.1007/s10858-021-00385-7","DOIUrl":"10.1007/s10858-021-00385-7","url":null,"abstract":"<div><p>Non-uniform sampling (NUS) is a popular way of reducing the amount of time taken by multidimensional NMR experiments. Among the various non-uniform sampling schemes that exist, the Poisson-gap (PG) schedules are particularly popular, especially when combined with compressed-sensing (CS) reconstruction of missing data points. However, the use of PG is based mainly on practical experience and has not, as yet, been explained in terms of CS theory. Moreover, an apparent contradiction exists between the reported effectiveness of PG and CS theory, which states that a “flat” pseudo-random generator is the best way to generate sampling schedules in order to reconstruct sparse spectra. In this paper we explain how, and in what situations, PG reveals its superior features in NMR spectroscopy. We support our theoretical considerations with simulations and analyses of experimental data from the Biological Magnetic Resonance Bank (BMRB). Our analyses reveal a previously unnoticed feature of many NMR spectra that explains the success of ”blue-noise” schedules, such as PG. We call this feature “clustered sparsity”. This refers to the fact that the peaks in NMR spectra are not just sparse but often form clusters in the indirect dimension, and PG is particularly suited to deal with such situations. Additionally, we discuss why denser sampling in the initial and final parts of the clustered signal may be useful.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00385-7.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4228439","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 : 2021-10-21DOI: 10.1007/s10858-021-00384-8
Tatsuya Matsunaga, Ryotaro Okabe, Yoshitaka Ishii
This study introduces a conceptually new solvent suppression scheme with adiabatic inversion pulses for 1H-detected multidimensional solid-state NMR (SSNMR) of biomolecules and other systems, which is termed “Solvent suppression of Liquid signal with Adiabatic Pulse” (SLAP). 1H-detected 2D 13C/1H SSNMR data of uniformly 13C- and 15N-labeled GB1 sample using ultra-fast magic angle spinning at a spinning rate of 60 kHz demonstrated that the SLAP scheme showed up to 3.5-fold better solvent suppression performance over a traditional solvent-suppression scheme for SSNMR, MISSISSIPPI (Zhou and Rienstra, J Magn Reson 192:167–172, 2008) with 2/3 of the average RF power.
{"title":"Efficient solvent suppression with adiabatic inversion for 1H-detected solid-state NMR","authors":"Tatsuya Matsunaga, Ryotaro Okabe, Yoshitaka Ishii","doi":"10.1007/s10858-021-00384-8","DOIUrl":"10.1007/s10858-021-00384-8","url":null,"abstract":"<div><p>This study introduces a conceptually new solvent suppression scheme with adiabatic inversion pulses for <sup>1</sup>H-detected multidimensional solid-state NMR (SSNMR) of biomolecules and other systems, which is termed “Solvent suppression of Liquid signal with Adiabatic Pulse” (SLAP). <sup>1</sup>H-detected 2D <sup>13</sup>C/<sup>1</sup>H SSNMR data of uniformly <sup>13</sup>C- and <sup>15</sup>N-labeled GB1 sample using ultra-fast magic angle spinning at a spinning rate of 60 kHz demonstrated that the SLAP scheme showed up to 3.5-fold better solvent suppression performance over a traditional solvent-suppression scheme for SSNMR, MISSISSIPPI (Zhou and Rienstra, J Magn Reson 192:167–172, 2008) with 2/3 of the average RF power.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4839561","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 : 2021-10-13DOI: 10.1007/s10858-021-00378-6
Stefano Cucuzza, Peter Güntert, Andreas Plückthun, Oliver Zerbe
{"title":"Correction to: An automated iterative approach for protein structure refinement using pseudocontact shifts","authors":"Stefano Cucuzza, Peter Güntert, Andreas Plückthun, Oliver Zerbe","doi":"10.1007/s10858-021-00378-6","DOIUrl":"10.1007/s10858-021-00378-6","url":null,"abstract":"","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00378-6.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4558299","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 : 2021-09-15DOI: 10.1007/s10858-021-00381-x
Young Kee Chae, Yoonjin Um, Hakbeom Kim
Protein-ligand interaction is one of the highlights of molecular recognition. The most popular application of this type of interaction is drug development which requires a high throughput screening of a ligand that binds to the target protein. Our goal was to find a binding ligand with a simple detection, and once this type of ligand was found, other methods could then be used to measure the detailed kinetic or thermodynamic parameters. We started with the idea that the ligand NMR signal would disappear if it was bound to the non-tumbling mass. In order to create the non-tumbling mass, we tried the aggregates of a target protein, which was fused to the elastin-like polypeptide. We chose the maltose binding proteinas a test case, and we tried it with several sugars, which included maltose, glucose, sucrose, lactose, galactose, maltotriose, and β-cyclodextrin. The maltose signal in the H-1 NMR spectrum disappeared completely as hoped around the protein to ligand ratio of 1:3 at 298 K where the proteins aggregated. The protein signals also disappeared upon aggregation except for the fast-moving part, which resulted in a cleaner background than the monomeric form. Since we only needed to look for a disappearing signal amongst those from the mixture, it should be useful in high throughput screening. Other types of sugars except for the maltotriose and β-cyclodextrin, which are siblings of the maltose, did not seem to bind at all. We believe that our system would be especially more effective when dealing with a smaller target protein, so both the protein and the bound ligand would lose their signals only when the aggregates formed. We hope that our proposed method would contribute to accelerating the development of the potent drug candidates by simultaneously identifying several binders directly from a mixture.
{"title":"A simple and sensitive detection of the binding ligands by using the receptor aggregation and NMR spectroscopy: a test case of the maltose binding protein","authors":"Young Kee Chae, Yoonjin Um, Hakbeom Kim","doi":"10.1007/s10858-021-00381-x","DOIUrl":"10.1007/s10858-021-00381-x","url":null,"abstract":"<div><p>Protein-ligand interaction is one of the highlights of molecular recognition. The most popular application of this type of interaction is drug development which requires a high throughput screening of a ligand that binds to the target protein. Our goal was to find a binding ligand with a simple detection, and once this type of ligand was found, other methods could then be used to measure the detailed kinetic or thermodynamic parameters. We started with the idea that the ligand NMR signal would disappear if it was bound to the non-tumbling mass. In order to create the non-tumbling mass, we tried the aggregates of a target protein, which was fused to the elastin-like polypeptide. We chose the maltose binding proteinas a test case, and we tried it with several sugars, which included maltose, glucose, sucrose, lactose, galactose, maltotriose, and β-cyclodextrin. The maltose signal in the H-1 NMR spectrum disappeared completely as hoped around the protein to ligand ratio of 1:3 at 298 K where the proteins aggregated. The protein signals also disappeared upon aggregation except for the fast-moving part, which resulted in a cleaner background than the monomeric form. Since we only needed to look for a disappearing signal amongst those from the mixture, it should be useful in high throughput screening. Other types of sugars except for the maltotriose and β-cyclodextrin, which are siblings of the maltose, did not seem to bind at all. We believe that our system would be especially more effective when dealing with a smaller target protein, so both the protein and the bound ligand would lose their signals only when the aggregates formed. We hope that our proposed method would contribute to accelerating the development of the potent drug candidates by simultaneously identifying several binders directly from a mixture.</p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10858-021-00381-x.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4631088","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 : 2021-09-12DOI: 10.1007/s10858-021-00382-w
Matthias Dreydoppel, Roman J. Lichtenecker, Mikael Akke, Ulrich Weininger
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 R1ρ 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 3JHH 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, kflip, agree well with previous results from 13C R1ρ 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 R1ρ relaxation dispersion experiment in combination with the site-selective 1H–13C/2H–12C labeling scheme enable measurement of exchange rates up to kex = 2kflip = 80,000 s–1, and serve as a useful complement to previously developed 13C-based methods.
{"title":"1H R1ρ relaxation dispersion experiments in aromatic side chains","authors":"Matthias Dreydoppel, Roman J. Lichtenecker, Mikael Akke, 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 <sup>1</sup>H <i>R</i><sub>1<i>ρ</i></sub> 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 <sup>1</sup>H–<sup>13</sup>C spin pairs with vicinal <sup>2</sup>H–<sup>12</sup>C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the <sup>3</sup><i>J</i><sub>HH</sub> couplings and limited chemical shift differences among <sup>1</sup>H 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><sub>flip</sub>, agree well with previous results from <sup>13</sup>C <i>R</i><sub>1<i>ρ</i></sub> relaxation dispersion experiments, and yield <sup>1</sup>H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic<sup>1</sup>H <i>R</i><sub>1<i>ρ</i></sub> relaxation dispersion experiment in combination with the site-selective <sup>1</sup>H–<sup>13</sup>C/<sup>2</sup>H–<sup>12</sup>C labeling scheme enable measurement of exchange rates up to <i>k</i><sub>ex</sub> = 2<i>k</i><sub>flip</sub> = 80,000 s<sup>–1</sup>, and serve as a useful complement to previously developed <sup>13</sup>C-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}
Pub Date : 2021-09-12DOI: 10.1007/s10858-021-00383-9
Zhiwei Miao, Qianqian Wang, Xiongjie Xiao, Ghulam Mustafa Kamal, Linhong Song, Xu Zhang, Conggang Li, Xin Zhou, Bin Jiang, Maili Liu
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.
{"title":"CSI-LSTM: a web server to predict protein secondary structure using bidirectional long short term memory and NMR chemical shifts","authors":"Zhiwei Miao, Qianqian Wang, Xiongjie Xiao, Ghulam Mustafa Kamal, Linhong Song, Xu Zhang, Conggang Li, Xin Zhou, Bin Jiang, 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}