complexes
NMR spectroscopy is a uniquely powerful technique, providing both structural and dynamic information on proteins at atomic resolution.
1.3.1
NMR of the 70S ribosome
As mentioned in Section 1.1.2, NMR spectroscopy has been applied to the study of 70S ribosomes, where the 15N HSQC spectrum of 70S ribosomes was found to contain a collection of resonances that arise almost exclusively from the L7/L12 stalk. The observation of the ribosomal protein L7/L12 is the result of the long, flexible linker that tethers the CTD to the ribosomal body. This allows the L7/L12 CTD to move almost independently of the ribosome, resulting in relatively narrow NMR signals despite its attachment to the large ribosomal body. The CTD domain is therefore capable of rapid tumbling and this is supported by15N relaxation studies, which have shown that the ribosome-bound L7/L12 CTD has a rotational correlation time of ca.14 ns [34], much shorter than that of the ribosome (ca. 2500 ns [122]).
Comparison of the chemical shifts of ribosome-bound L7/L12 compared with those obtained from isolated L7/L12 reveals close similarities between the two species despite their different physical environments [34]. While this strongly indicates that L7/L12 can acquire the equivalent structure on the ribosome as in isolation, its dynamic behaviour on the
ribosome remains to be explored in detail. Moreover, detailed structural information in the form of its orientation and spatial positioning on the ribosome is currently unavailable, and the development of NMR strategies that seek to describe these characteristics is described in Chapter 2.
1.3.2
NMR studies of RNCs
The use of NMR spectroscopy to inspect highly mobile regions on the ribosome, such as L7/L12, provided the motivation for an initial study into the possibility of applying NMR to ribosome-bound nascent chains. This approach was first demonstrated for a pair of tandem-repeat immunoglobulin-like domains (domains 5 and 6) derived from the F-actin cross-linking gelation factor from Dictyostelium discoideum, referred to in this study as ddFLN646-838. An RNC was generated for ddFLN646-838 in which the folding incompetent domain 6 formed a flexible linker that tethered domain 5 to the PTC, a scenario reminiscent of ribosome-associated L7/L12.
The use of rapid acquisition methods such as the SOFAST-HMQC experiment [123] on uniformly 15N-labelled RNCs allowed observation of 15N HMQC spectra, which showed that domain 5 adopted a native fold that resembled the structure of the isolated protein. The folded state has since been further studied using1H-13C correlation spectra, exploiting the enhanced sensitivity afforded by the methyl-containing side-chains [124].
Similar NMR approaches have been applied in the study of barnase-RNCs [125] and also SH3-RNCs using a 1H-15N CRINEPT strategy [126]. The latter revealed that a disordered SH3 domain was observable whilst bound the ribosome, but could acquire its native structure upon release. Together these studies have allowed initial structural and dynamic characterisation of ribosome-bound NCs. However, at present, direct structural information on the nascent chain is limited.
1.3.3
Use of translational diffusion to monitor ribosomal integrity
By virtue of its inherently dynamic nature, the ribosome has a limited lifetime in vitro. An important NMR tool to evaluate the integrity of the ribosome is translational diffusion. The use of translational diffusion in this manner follows from extensive studies of the diffusional properties of many isolated proteins [127].
The translational diffusion of a molecule is directly related to its hydrodynamic radius according to the Stokes-Einstein equation:
D= kBT
6πηr (1.1)
where D is the diffusion coefficient calculated from the diffusion experiment, η is the viscosity of the solution, r is the radius of the spherical particle, T is the temperature and kB is Bolzmann’s constant.
The hydrodynamic radius calculated using the Stokes-Einstein equation can then be used to estimate the molecular weight according to the following equation:
M = 1 v · 4 3π rh−δ fe/ fo !3 · NA (1.2)
where M is the molecular weight, v is the specific volume, rhis the hydrodynamic radius, δ is the thickness of the hydrated water layer, fe/ fo is the shape factor and NA is Avogadro’s number.
In this calculation, there are two principal assumptions. Firstly, the thickness of the hydrated water layer is assumed to correspond to a single layer of water molecules, but this parameter is difficult to be determined accurately. Secondly, the shape factor is usually assumed to be 1, as for a perfectly spherical globular protein. This shape factor can be calculated according to the shape of the protein, if it is known.
The diffusion constant is measured using a gradient spin echo experiment. In its simplest form, this experiment consists of an excitation pulse followed by two gradients of opposite sign, separated by a delay. The first gradient acts to encode the position of the spins as a
spatially dependent phase factor. This spatially dependent phase is decoded by the second gradient, but this decoding will only be perfect if there is no movement of spins between the gradients. In practice, this condition is violated due to diffusion-mediated molecular motion during the inter-gradient delay. This leads to imperfect refocusing of the spatially dependent phase, and hence a decay in signal intensity. The decay of signal intensity is described by the Stejskal-Tanner equation [128]:
I = I0· exp
−D(γδG)2(∆ −δ/3
(1.3)
The observed intensity, I, depends on the initial intensity I0, the diffusion constant D, the length δ and strength G of the encode/decode gradients, the inter-gradient diffusion delay ∆ and the magnetogyric ratio γ of the detected spin.
In this work, diffusion experiments are used mostly to monitor the integrity of the ribosome complex and the attachment of the NC. A fresh, intact ribosome sample shows slow diffusion with a diffusion coefficient of 1.7-2x10-11m2s-1. Small proteins have diffusion coefficients on the order of 1x10-10m2s-1. While the NC is attached to the ribosome, the translational diffusion of the NC is the same as the ribosome. But when measuring simple 1H diffusion experiments, the detected signal is dominated by the ribosome as the ribosomal signals swamp those from the NC. To distinguish between the NC and the ribosome, a15N- edited diffusion experiment is used, which only detects signals from 15N-labelled species (the NC). The analysis of diffusion data is important in the study of ribosomes and RNCs, as the integrity of the ribosome and attachment of the NC is crucial for reliable analysis and interpretation of the NMR data.