Arnout P Kalverda, James Gowdy, Gary S Thompson, Steve W Homans, Peter J F Henderson, Simon G Patching
{"title":"TROSY核磁共振与52 kDa糖转运蛋白和结合的小分子抑制剂。","authors":"Arnout P Kalverda, James Gowdy, Gary S Thompson, Steve W Homans, Peter J F Henderson, Simon G Patching","doi":"10.3109/09687688.2014.911980","DOIUrl":null,"url":null,"abstract":"<p><p>Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"31 4","pages":"131-40"},"PeriodicalIF":0.0000,"publicationDate":"2014-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2014.911980","citationCount":"13","resultStr":"{\"title\":\"TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.\",\"authors\":\"Arnout P Kalverda, James Gowdy, Gary S Thompson, Steve W Homans, Peter J F Henderson, Simon G Patching\",\"doi\":\"10.3109/09687688.2014.911980\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.</p>\",\"PeriodicalId\":18858,\"journal\":{\"name\":\"Molecular Membrane Biology\",\"volume\":\"31 4\",\"pages\":\"131-40\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2014-06-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.3109/09687688.2014.911980\",\"citationCount\":\"13\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Membrane Biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.3109/09687688.2014.911980\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2014/5/7 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Membrane Biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3109/09687688.2014.911980","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2014/5/7 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.
Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.
期刊介绍:
Cessation.
Molecular Membrane Biology provides a forum for high quality research that serves to advance knowledge in molecular aspects of biological membrane structure and function. The journal welcomes submissions of original research papers and reviews in the following areas:
• Membrane receptors and signalling
• Membrane transporters, pores and channels
• Synthesis and structure of membrane proteins
• Membrane translocation and targeting
• Lipid organisation and asymmetry
• Model membranes
• Membrane trafficking
• Cytoskeletal and extracellular membrane interactions
• Cell adhesion and intercellular interactions
• Molecular dynamics and molecular modelling of membranes.
• Antimicrobial peptides.