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Evaluation of the tert-butyl group as a probe for NMR studies of macromolecular complexes
- Source :
- Digital.CSIC. Repositorio Institucional del CSIC, instname, J Biomol NMR
- Publication Year :
- 2021
- Publisher :
- Springer, 2021.
-
Abstract
- 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 200 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.<br />We thank Ad Bax for the suggestion of exploring the use tBu groups as probes for structural studies of the neurotransmitter release machinery, and Ad Bax, Lewis Kay and Charampalos Kalodimos for fruitful discussions on this subject. The Agilent DD2 console of the 800 MHz spectromenter used for the research presented here was purchased with a shared instrumentation grant from the NIH (S10OD018027 to JR). Rashmi Voleti was supported by a fellowship from the Howard Hughes Medical Institute. The preparation of BDSNB was performed at the NANBIOSIS –CIBER BBN Peptide Synthesis Unit (U3). This work was supported by grant I-1304 from the Welch Foundation (to JR) and by NIH Research Project Award R35 NS097333 (to JR).
- Subjects :
- Magnetic Resonance Spectroscopy
Macromolecular Substances
Stereochemistry
Complexin
Biochemistry
Article
SNARE complex
chemistry.chemical_compound
Transverse relaxation-optimized spectroscopy
Solubility
Nuclear Magnetic Resonance, Biomolecular
Spectroscopy
Neurons
Neurotransmitter release
Nuclear magnetic resonance spectroscopy
Tert-butyl NMR
Synaptotagmin
Membrane
Macromolecular assemblies
chemistry
Iodoacetamide
Proton NMR
Slow relaxation
SNARE Proteins
Macromolecule
Cysteine
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Digital.CSIC. Repositorio Institucional del CSIC, instname, J Biomol NMR
- Accession number :
- edsair.doi.dedup.....a07aa754454b0f554cc737872e978274