The development of methyl transverse relaxation optimized spectroscopy has greatly facilitated the study of macromolecular assemblies by solution NMR spectroscopy. However, limited sample solubility and stability has hindered application of this technique to ongoing studies of complexes formed on membranes by the neuronal SNAREs that mediate neurotransmitter release and synaptotagmin-1, the Ca2+ sensor that triggers release. Since the 1H NMR signal of a tBu group attached to a large protein or complex can be observed with high sensitivity if the group retains high mobility, we have explored the use of this strategy to analyze presynaptic complexes involved in neurotransmitter release. For this purpose, we attached tBu groups at single cysteines of fragments of synaptotagmin-1, complexin-1 and the neuronal SNAREs by reaction with 5-(tert-butyldisulfaneyl)-2-nitrobenzoic acid (BDSNB), tBu iodoacetamide or tBu acrylate. The tBu resonances of the tagged proteins were generally sharp and intense, although tBu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the tBu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the tBu resonances in some cases. However, sharp tBu resonances could readily be observed for some complexes of more than 200Â*kDa at low micromolar concentrations. Our results show that tagging of proteins with tBu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations.
[NMR paper] Perfluoro-tert-Butyl Hydroxyprolines as Sensitive, Conformationally Responsive Molecular Probes: Detection of Protein Kinase Activity by 19F NMR.
Perfluoro-tert-Butyl Hydroxyprolines as Sensitive, Conformationally Responsive Molecular Probes: Detection of Protein Kinase Activity by 19F NMR.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-pubmed-acspubs.jpg Related Articles Perfluoro-tert-Butyl Hydroxyprolines as Sensitive, Conformationally Responsive Molecular Probes: Detection of Protein Kinase Activity by 19F NMR.
ACS Chem Biol. 2020 Mar 03;:
Authors: Tressler CM, Zondlo NJ
Abstract
19F NMR spectroscopy provides the ability to...
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[NMR paper] Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy.
Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy.
Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy.
J Biomol NMR. 2017 Dec 02;:
Authors: Loh CT, Adams LA, Graham B, Otting G
Abstract
The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and...
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Genetically encoded amino acids with tert -butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy
Genetically encoded amino acids with tert -butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy
Abstract
The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporated into different proteins, using an amber stop codon, suppressor tRNA and the broadband aminoacyl-tRNA synthetase originally evolved for the incorporation of p-cyano-phenylalanine. The 1H-NMR signals of...
[NMR paper] (2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4-hydroxyproline: two conformationally distinct proline amino acids for sensitive application in 19F NMR.
(2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4-hydroxyproline: two conformationally distinct proline amino acids for sensitive application in 19F NMR.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-pubmed-acspubs.jpg Related Articles (2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4-hydroxyproline: two conformationally distinct proline amino acids for sensitive application in 19F NMR.
J Org Chem. 2014 Jun 20;79(12):5880-6
Authors: Tressler CM, Zondlo NJ
Abstract
(2S,4R)- and...
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[NMR paper] Perfluoro-tert-butyl-homoserine as a sensitive (19) F NMR reporter for peptide-membrane interactions in solution.
Perfluoro-tert-butyl-homoserine as a sensitive (19) F NMR reporter for peptide-membrane interactions in solution.
Related Articles Perfluoro-tert-butyl-homoserine as a sensitive (19) F NMR reporter for peptide-membrane interactions in solution.
J Pept Sci. 2013 Mar 19;
Authors: Buer BC, Levin BJ, Marsh EN
Abstract
Fluorine ((19) F) NMR is a valuable tool for studying dynamic biological processes. However, increasing the sensitivity of fluorinated reporter molecules is a key to reducing acquisition times and accessing transient...
Evaluation of the tert-butyl group as a probe for NMR studies of macromolecular complexes
Linear Mode
