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

J Biomol NMR (2015) 63:151–163 DOI 10.1007/s10858-015-9968-x ARTICLE Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome–nascent chain complexes Sammy H. S. Chan1 • Christopher A. Waudby1 • Anaı̈s M. E. Cassaignau1 • Lisa D. Cabrita1 • John Christodoulou1 Received: 31 May 2015 / Accepted: 13 July 2015 / Published online: 8 August 2015 Ó The Author(s) 2015. This article is published with open access at Springerlink.com 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 lM), 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 Nz during diffusion delays or introducing significant transverse relaxation line broadening. By applying the longitudinal relaxation-optimized SORDID pulse Sammy H. S. Chan and Christopher A. Waudby have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10858-015-9968-x) contains supplementary material, which is available to authorized users. & John Christodoulou 1 Institute of Structural and Molecular Biology, University College London and Birkbeck College, London WC1E 6BT, UK 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. Graphical Abstract O ribosome-nascent chain complex - H N N 4.5-fold increase in sensitivity of NMR diffusion measurements O Ni 2+ O- N N H O in combination with longitudinal relaxation-enhanced pulse sequence 10 8 H / ppm 6 10 8 H 6 / ppm Keywords Diffusion NMR spectroscopy  Paramagnetic longitudinal relaxation enhancement  Ribosome–nascent chain complex  NMR sensitivity enhancement Introduction NMR diffusion measurements are a powerful probe of biomolecular structure and dynamics in which the translational properties of molecules can be examined non-invasively, using a very wide variety of gradient echo NMR 123 152 experiments (Johnson 1999). These measurements can be used to determine diffusion coefficients, which in turn can be related to hydrodynamic radii and hence molecular structure by the Stokes–Einstein equation. The development of NMR diffusion methods has thereby advanced studies in a wide range of areas in biology, such as the analysis of peptide aggregation and amyloid formation (Baldwin et al. 2008); macromolecular crowding effects (Li et al. 2009); protein–ligand binding events (Lucas and Larive 2004); and in-cell NMR to distinguish between intra- and extracellular proteins (Waudby et al. 2012). Furthermore, NMR measurements of diffusion have been used to investigate how secondary structure and hydrophobic clusters affect the hydrodynamic radii within different conformational ensembles including partially folded and molten globule states (Wilkins et al. 1999). With increasing applications of NMR spectroscopy in understanding the biology of complex systems, NMR diffusion measurements are likely to develop growing prominence. The measurement of translational diffusion has also played an important role in NMR studies of large macromolecular assemblies, including investigations of ribosomal particles (Christodoulou et al. 2004; Cabrita et al. 2009; Hsu et al. 2007; Eichmann et al. 2010). The study of such complexes is of major biological interest, but the high molecular weight and the resulting low maximum achievable concentrations, most often combined with limited sample lifetimes, commonly results in very weak signals that present significant spectroscopic challenges (Waudby et al. 2013). An example of this is seen in recent studies of ribosome–bound nascent chain complexes (RNCs), in which sample lifetimes are limited primarily by release of the nascent chain from the ribosome before degradation of the ribosome itself (Waudby et al. 2013). The continuous monitoring of translational diffusion is therefore essential to ensure that the observed resonances arise from an intact complex. In particular, isotope-edited diffusion experiments, and especially the heteronuclear stimulated-echo (XSTE) experiment (Ferrage et al. 2003) have been critical in allowing the attachment of the isotopically-labelled nascent chain to the (unlabeled) ribosome to be monitored specifically (Cabrita et al. 2009; Hsu et al. 2007; Eichmann et al. 2010; Waudby et al. 2013), an approach similar to one first used to study the dynamic regions of free ribosomes (Christodoulou et al. 2004). Given such constraints to NMR studies of ribosomal particles, continual improvements in experimental sensitivity are central to progress in this field. Large gains in sensitivity and resolution have been made through the availability of high-field spectrometers (Rovnyak et al. 2004) and cryogenic probes (Kovacs et al. 2005). Transverse relaxation optimized spectroscopy (TROSY) (Pervushin et al. 1997; Fernández and Wider 2003) and methyl- 123 J Biomol NMR (2015) 63:151–163 TROSY (Tugarinov et al. 2003) in combination with advanced isotopic labeling schemes (Tugarinov et al. 2006) have revolutionised the study of large systems by NMR spectroscopy, such as the 900 kDa GroEL–GroES complex (Fiaux et al. 2002) and 670 kDa 20S proteasome (Sprangers and Kay 2007), and these methods are beginning to be applied to the study of RNCs (Eichmann et al. 2010). Furthermore, other techniques such as non-uniform (sparse) sampling (Hyberts et al. 2012) or non-uniform weighted sampling (Waudby and Christodoulou 2012) may also further contribute to sensitivity improvements in multi-dimensional NMR by sampling more efficiently on the Nyquist grid. In typical NMR measurements, the majority of spectrometer time ([90 %) is (...truncated)