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. 2015 Oct;63(2):151-163.
doi: 10.1007/s10858-015-9968-x. Epub 2015 Aug 8.

Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes

Sammy H S Chan #  1 , Christopher A Waudby #  1 , Anaïs M E Cassaignau  1 , Lisa D Cabrita  1 , John Christodoulou  1
Affiliations

Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes

Sammy H S Chan et al. J Biomol NMR. 2015 Oct.

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 (15)N 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 (1)H 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.

Keywords: Diffusion NMR spectroscopy; NMR sensitivity enhancement; Paramagnetic longitudinal relaxation enhancement; Ribosome–nascent chain complex.

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Figures

Fig. 1
Fig. 1
The effect of paramagnetic longitudinal relaxation enhancement on the sensitivity of NMR measurements of a–c ddFLN5 and d–f α-synuclein. Overlay of 2D 1H–15N SOFAST-HMQC spectra a, d of isolated proteins in the presence and absence of 40 mM NiDO2; insets show magnified views of highlighted resonances. The relative sensitivity of b, e 1D 1H–15N SOFAST-HMQC experiments as a function of the recovery delay Trec, and of c, f XSTE and SORDID diffusion experiments as a function of the diffusion delay Δ. Data are fitted to their theoretical sensitivity expressions (Eqs. 8, 9)
Fig. 2
Fig. 2
The effect of paramagnetic longitudinal relaxation enhancement on the sensitivity of NMR measurements of E. coli 70S ribosomes. a Overlay of 2D 1H–15N SOFAST-HMQC spectra in the presence and absence of 40 mM NiDO2A PLRE agent. Dashed lines indicate locations of one-dimensional slices as shown in b, c, d and labeled with known assignments from the L7/L12 stalk complex. The relative sensitivity of e 1D 1H–15N SOFAST-HMQC experiments as a function of the recovery delay Trec, and of f XSTE and SORDID diffusion experiments as a function of the diffusion delay Δ. Data are fitted to their theoretical sensitivity expressions (Eqs. 8, 9)
Fig. 3
Fig. 3
The effect of paramagnetic longitudinal relaxation enhancement on the sensitivity of SOFAST-HMQC experiments of a ddFLN5 RNC. Overlay of a 1D 1H–15N SOFAST-HMQC spectra and of b 2D 1H–15N SOFAST-HMQC spectra (of 30 min experimental time each) in the presence and absence of 40 mM NiDO2A. Additional resonances are observable in the PLRE-induced spectrum and all can be assigned to the nascent chain or the L7/L12 stalk region of background-labeled ribosomes. Those that can be assigned unambiguously to either region are circled in red or blue respectively, and labeled with their known assignments. Dashed lines indicate locations of one-dimensional slices as shown in c, d, e and labeled with known and unambiguous assignments of unfolded ddFLN5 or L7/12
Fig. 4
Fig. 4
Comparison of NMR diffusion measurements of a ddFLN5 RNC, and monitoring its integrity using both biochemical and NMR analysis. a XSTE (Δ = 100 ms; G = 5 %, 95 % Gmax) and SORDID (Δ = 190 ms; G = 10.4 %, 69.5 % Gmax) spectra acquired within 30 min each, in the presence and absence of 40 mM NiDO2A. b Relative sensitivity for each diffusion experiment. c Diffusion coefficients measured by each diffusion experiment. d Diffusion coefficients of the RNC sample measured by sets of XSTE and SORDID experiments to examine the integrity and stability of the RNC. e Western blot analysis to assess the effect of 40 mM NiDO2A on the RNC integrity, detected using anti-His (upper) and anti-SecM (lower) antibodies. Upshifted bands (~40 kDa) correspond to the tRNA-bound form of the RNC in which the nascent chain is attached to the ribosome, with release of the nascent chain (and tRNA) monitored by the band at a lower molecular weight of ~23 kDa
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