Abstract
NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome–nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity.
Highlights
The process by which a nascent polypeptide acquires its native folded structure after emerging from the ribosomal exit tunnel has been the subject of intense research since the pioneering observations of Anfinsen and Levinthal [1,2,3,4,5,6]
It is here that NMR spectroscopy has proved to be a highly complementary technique, for the flexibility of such regions results in a reduction in their effective rotational correlation time, giving rise to sharp resonances despite the large size of the core ribosome particle. This observation led in 2004 to the high-resolution characterization of the structure and dynamics of the L7/L12 ribosome stalk region [54,55], which due to its flexibility was not observable in crystallographic studies of the ribosome. These initial findings stimulated the investigation of the structural and dynamical properties of ribosome-bound nascent chains during co-translational folding by NMR and, in 2007, we reported the first observation of a ribosome–nascent chain complex (RNC) by solution-state NMR spectroscopy [36]
NMR studies of the isolated L7/12 protein subsequently identified a conserved region on the surface of the C-terminal domain that interacts with EF-G, and initiation factor 2 (IF2) and elongation factor Tu (EF-Tu), with millimolar affinity which, given the high local concentration of these translation factors when bound to the ribosome, results in large populations of the bound state on the ribosome [65]
Summary
The process by which a nascent polypeptide acquires its native folded structure after emerging from the ribosomal exit tunnel has been the subject of intense research since the pioneering observations of Anfinsen and Levinthal [1,2,3,4,5,6]. It is here that NMR spectroscopy has proved to be a highly complementary technique, for the flexibility of such regions results in a reduction in their effective rotational correlation time, giving rise to sharp resonances despite the large size of the core ribosome particle This observation led in 2004 to the high-resolution characterization of the structure and dynamics of the L7/L12 ribosome stalk region [54,55], which due to its flexibility was not observable in crystallographic studies of the ribosome. We will initially review the use of solution-state NMR spectroscopy for the study of the structure and dynamics of isolated ribosomes (Section 2) These observations were instrumental in demonstrating the feasibility of high-resolution studies of flexible domains within very large molecules, and led directly to studies of ribosome–nascent chain complexes by NMR. We close by discussing the potential for the field in the future (Section 5)
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