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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 Ca 2+ sensor that triggers release. Since the 1 H NMR signal of a t Bu 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 t Bu 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), t Bu iodoacetamide or t Bu acrylate. The t Bu resonances of the tagged proteins were generally sharp and intense, although t Bu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the t Bu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the t Bu resonances in some cases. However, sharp t Bu 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 t Bu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations.

Evaluation of the tert-butyl group as a probe for NMR studies of macromolecular complexes