Yi Jiun Tan, Elwy H. Abdelkader, Eliza Tarcoveanu, Ansis Maleckis, Christoph Nitsche and Gottfried Otting*,
{"title":"大肠杆菌 PpiB 中的 (2S,4S)-5-氟亮氨酸、(2S,4R)-5-氟亮氨酸和 5,5'-二氟亮氨酸:通过 γ-Gauche 效应增强的蛋白质生成、19F NMR 和配体感应。","authors":"Yi Jiun Tan, Elwy H. Abdelkader, Eliza Tarcoveanu, Ansis Maleckis, Christoph Nitsche and Gottfried Otting*, ","doi":"10.1021/acs.biochem.4c00080","DOIUrl":null,"url":null,"abstract":"<p >Global substitution of leucine for analogues containing CH<sub>2</sub>F instead of methyl groups delivers proteins with multiple sites for monitoring by <sup>19</sup>F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa <i>Escherichia coli</i> peptidyl–prolyl <i>cis–trans</i> isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2<i>S</i>,4<i>S</i>)-5-fluoroleucine, (2<i>S</i>,4<i>R</i>)-5-fluoroleucine, or 5,5′-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. <sup>19</sup>F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The <sup>19</sup>F chemical shifts correlate with the three-bond <sup>1</sup>H–<sup>19</sup>F couplings (<sup>3</sup><i>J</i><sub>HF</sub>), providing the first experimental verification of the γ-gauche effect predicted by [<contrib-group><span>Feeney, J.</span></contrib-group> <cite><i>J. Am. Chem. Soc.</i></cite> <span>1996</span>, <em>118</em>, 8700–8706] and establishing the effect as the predominant determinant of the <sup>19</sup>F chemical shifts of CH<sub>2</sub>F groups. Individual CH<sub>2</sub>F groups can be confined to single rotameric states by the protein environment, but most CH<sub>2</sub>F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5′-difluoroleucine bias the CH<sub>2</sub>F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the <sup>19</sup>F chemical shift to the rotameric state of the CH<sub>2</sub>F groups potentially renders them particularly sensitive for detecting allosteric effects.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5′-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect\",\"authors\":\"Yi Jiun Tan, Elwy H. Abdelkader, Eliza Tarcoveanu, Ansis Maleckis, Christoph Nitsche and Gottfried Otting*, \",\"doi\":\"10.1021/acs.biochem.4c00080\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Global substitution of leucine for analogues containing CH<sub>2</sub>F instead of methyl groups delivers proteins with multiple sites for monitoring by <sup>19</sup>F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa <i>Escherichia coli</i> peptidyl–prolyl <i>cis–trans</i> isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2<i>S</i>,4<i>S</i>)-5-fluoroleucine, (2<i>S</i>,4<i>R</i>)-5-fluoroleucine, or 5,5′-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. <sup>19</sup>F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The <sup>19</sup>F chemical shifts correlate with the three-bond <sup>1</sup>H–<sup>19</sup>F couplings (<sup>3</sup><i>J</i><sub>HF</sub>), providing the first experimental verification of the γ-gauche effect predicted by [<contrib-group><span>Feeney, J.</span></contrib-group> <cite><i>J. Am. Chem. Soc.</i></cite> <span>1996</span>, <em>118</em>, 8700–8706] and establishing the effect as the predominant determinant of the <sup>19</sup>F chemical shifts of CH<sub>2</sub>F groups. Individual CH<sub>2</sub>F groups can be confined to single rotameric states by the protein environment, but most CH<sub>2</sub>F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5′-difluoroleucine bias the CH<sub>2</sub>F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the <sup>19</sup>F chemical shift to the rotameric state of the CH<sub>2</sub>F groups potentially renders them particularly sensitive for detecting allosteric effects.</p>\",\"PeriodicalId\":28,\"journal\":{\"name\":\"Biochemistry Biochemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.9000,\"publicationDate\":\"2024-05-16\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochemistry Biochemistry\",\"FirstCategoryId\":\"1\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/acs.biochem.4c00080\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry Biochemistry","FirstCategoryId":"1","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.biochem.4c00080","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5′-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect
Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl–prolyl cis–trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5′-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H–19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J.J. Am. Chem. Soc.1996, 118, 8700–8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5′-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.
期刊介绍:
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