Chan, Sammy H.S. and Waudby, Christopher A. and Cassaignau, Anais M.E. and Cabrita, Lisa D. and Christodoulou, John (2015) Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome–nascent chain complexes. Journal of Biomolecular NMR 63 (2), pp. 151-163. ISSN 0925-2738.
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Abstract
The translational diffusion of macromolecules can be examined non-invasively by stimulated echo (STE) NMR experiments to accurately determine their molecular sizes. These measurements can be important probes of intermolecular interactions and protein folding and unfolding, and are crucial in monitoring the integrity of large macromolecular assemblies such as ribosome–nascent chain complexes (RNCs). However, NMR studies of these complexes can be severely constrained by their slow tumbling, low solubility (with maximum concentrations of up to 10 μM), and short lifetimes resulting in weak signal, and therefore continuing improvements in experimental sensitivity are essential. Here we explore the use of the paramagnetic longitudinal relaxation enhancement (PLRE) agent NiDO2A on the sensitivity of 15N XSTE and SORDID heteronuclear STE experiments, which can be used to monitor the integrity of these unstable complexes. We exploit the dependence of the PLRE effect on the gyromagnetic ratio and electronic relaxation time to accelerate recovery of 1H magnetization without adversely affecting storage on N z during diffusion delays or introducing significant transverse relaxation line broadening. By applying the longitudinal relaxation-optimized SORDID pulse sequence together with NiDO2A to 70S Escherichia coli ribosomes and RNCs, NMR diffusion sensitivity enhancements of up to 4.5-fold relative to XSTE are achieved, alongside ~1.9-fold improvements in two-dimensional NMR sensitivity, without compromising the sample integrity. We anticipate these results will significantly advance the use of NMR to probe dynamic regions of ribosomes and other large, unstable macromolecular assemblies.
Metadata
| Item Type: | Article |
|---|---|
| Keyword(s) / Subject(s): | Diffusion NMR spectroscopy, Paramagnetic longitudinal relaxation enhancement, Ribosome–nascent chain complex, NMR sensitivity enhancement |
| School: | Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences |
| Research Centres and Institutes: | Structural Molecular Biology, Institute of (ISMB) |
| Depositing User: | Administrator |
| Date Deposited: | 10 Aug 2015 12:14 |
| Last Modified: | 02 Aug 2023 17:18 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/12743 |
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