Structural investigation of co-translational folding events on the ribosome by NMR spectroscopy

Lead Research Organisation: University College London
Department Name: Structural Molecular Biology

Abstract

The human body is likely to contain more than 2 million proteins, and every function in every living cell depends critically on them. Proteins are made up of building blocks called amino acids, which are linked together and arranged in various combinations (the sequence) to make a chain. The sequence results in a unique protein capable of a different function inside the body. In all kingdoms of life, protein manufacture by the cell occurs by a process known as translation and is carried out by highly sophisticated, miniature factories called ribosomes - these 'machines' are able to decode the instructions contained within our genetically inherited DNA blueprint and build a protein by adding amino acids one at a time to form the chain. During manufacture, the newly made chain, or 'nascent chain' (NC), journeys from the epicentre of these ribosome particles through a protective passage known as the ribosomal tunnel and then into the hostile cellular environment. This NC thus tries to protect itself so rather than resembling an extended string, it attempts to wrap up or 'fold' into its characteristic shape; the shape gives the protein a different function. The information which directs the NC folding is contained within the amino acid sequence. However, how this sequence provides instructions for folding is a central question in biology and it is indeed still one of the most hotly contested 'holy grails' of science. It is critical for a protein to adopt its shape quickly and efficiently in the cell. Should a protein chain have the wrong coding in its sequence it could fold into the incorrect structure and the consequences could be devastating, leading to diseases such as Type II Diabetes, Alzheimer's and Parkinson's, vCJD (Mad Cow disease), cystic fibrosis and many others. An understanding of how a protein folds, will allow us to try and reverse or prevent the occasions when it misfolds. At present, most studies have examined the folding and three-dimensional structure and mobility of a protein while it is in a test tube after its production by the ribosome. While this has given some incredible insights into protein behaviour, very little is known about how this process occurs within the cell. Our interest is centred on looking at the protein chain as it is being made on its ribosome. We propose to take high-resolution snapshots of the manufacture of a protein chain as it progressively exits the ribosome so that we can understand its development. A part of our proposal is geared towards developing and implementing emerging technologies to produce these snapshots - we will use the tools of genetic engineering to program the ribosomes within bacterial cells to halt at different stages during protein manufacture. This will be followed by a strategy to remove the ribosomes from the cells and into the test tube, after which we can analyze the sample using a powerful visual technique called Nuclear Magnetic Resonance (NMR), which can examine proteins at the level of the atom. The aim is to take many snapshots of protein chains of different lengths and showing a dynamic slideshow of the manufacturing process - the series of events that takes place from the time the protein chain leaves the ribosome until it forms its structure. We also plan to use these snapshots to understand what the ribosome looks like as it is making a new protein. This ambitious and challenging plan will mean that, for the first time and in extraordinary detail, we will be able to describe how a protein forms its structure when it is being made in the cell, a significant step closer to understanding protein folding in its natural environment and make inroads into why the misfolding processes described take place. This level of structural knowledge can also be used to rationally design small molecules (drugs) that can bind to a protein of interest and prevent any misfolding of the protein and as a consequence prevent the onset of disease states.

Technical Summary

This proposal aims to build on our recent success in showing that we can structurally analyze a protein chain while it is being created biosynthetically. It seeks to understand, at a detailed structural and dynamical level, the co-translational folding processes of ribosome-bound nascent chains. The structural biology of the ribosome has progressed very significantly over recent years although the structural details of an attached nascent chain (NC) have been elusive. The aim proposed here is to use the complementary properties of NMR spectroscopy which can uniquely provide detailed structural information on dynamically disordered states, in order to delineate the conformational properties (at high resolution and at a residue specific level) of emerging NCs stalled during protein synthesis. This work presents an ideal opportunity for unprecedented insights into the ribosome and of protein folding at the level of synthesis and as a result, it thus has potential applications to the many areas that protein folding critically influences such as translocation and degradation, to understanding protein misfolding and the formation of multimeric complexes, as well as in the design of novel antibiotics.

Publications

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Waudby CA (2011) GPU accelerated Monte Carlo simulation of pulsed-field gradient NMR experiments. in Journal of magnetic resonance (San Diego, Calif. : 1997)

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O'Brien EP (2011) New scenarios of protein folding can occur on the ribosome. in Journal of the American Chemical Society

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O'Brien EP (2010) Transient tertiary structure formation within the ribosome exit port. in Journal of the American Chemical Society

 
Description Our research examines the earliest events of how proteins are manufactured inside cells; specifically we study how proteins acquire their shape, as this is important for how proteins carry out their function. In addition, when proteins are unable to acquire their shape, they can form toxic clumps inside cells and lead to a number of diseases.

Our research has involved developing and applying a powerful visual technique, NMR spectroscopy to study proteins as they are being made by ribosomes, the cellular machines responsible for protein production. To enable us to study proteins and the ribosome in this way, we have also developed genetic tools in E.coli cells, which is essentially a well-studied factory.

Using genetic tools, we have developed ways to programme the ribosomes within E.coli cells to produce proteins on demand, and in this way, we have created "snapshots" of the protein production process, enabling us to use NMR spectroscopy along with computational calculations, to study the molecular details of how the protein forms its shape.

With this, we have been able to produce a three-dimensional picture of what a protein looks like as it is being manufactured, and in addition we are beginning to observe the influence of the ribosome during this process. This has important implications for understanding how proteins form their shapes; we can use these studies as a basis for designing drugs that can prevent diseases: for example, by diverting proteins towards forming alternative shapes, if they are likely to otherwise form toxic clumps, or by targeting the ribosome itself - an important route for identifying new antibiotics.
Exploitation Route Our research has enabled us to develop novel NMR and biochemical technologies to study protein biosynthesis, and in particular to provide high-resolution information of complex molecular machines and of protein structure and dynamics. The molecular details that are emerging from our research, offer novel structural considerations for how protein structure formation in the cell is understood, as well as how the ribosome is involved in this process. This contributes to an understanding of a fundamental molecular mechanism underpinning the activity of all cells, and is a key means of developing novel drug targets.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Wellcome Trust
Amount £1,000,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 09/2017
 
Title An analysis of NMR sensitivity enhancements obtained using non-uniform weighted sampling 
Description We have explored strategies using non-uniform weighted sampling during NMR acquisition, as an important addition to rapid acquisition techniques. This approach provides a means of studying challenging dynamic systems. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact N/A 
 
Title Generation of ribosome-nascent chain complexes 
Description We have developed a strategy in E.coli to generate isotopically-labelled ribosome-nascent chain complexes for the study of co-translational protein folding using NMR spectroscopy 
Type Of Material Biological samples 
Year Produced 2010 
Provided To Others? Yes  
Impact This strategy has initiated structural studies of ribosome-nascent chain complexes of other protein systems. 
 
Title Increasing the sensitivity of NMR diffusion experiments by paramagnetic longitudinal relaxation enhancement 
Description This is a tool that enables the enhancement of signals arising from NMR translational diffusion experiments, which are key experiments used to determine the molecular sizes of biomolecules. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact N/A 
 
Title NMR spectroscopy of ribosome-nascent chain complexes 
Description We have developed NMR strategies to study ribosome-nascent chain complexes. Specifically, we have designed a suite of NMR experiments that enable the parallel study of the structure and dynamics of nascent chain, as well as the integrity of the complex. The latter is particularly important for ensuring that the NMR studies reflect ribosome-attached nascent chains. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact We have been approached by several groups to begin NMR studies of co-translational folding of their systems. This work is ongoing. 
 
Description NMR-restrained molecular dynamics simulations 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution From our structural studies of ribosome-nascent chain complexes, we have generated NMR data, in the form of chemical shift information, and residual dipolar couplings, which are used as inputs for molecular dynamics simulations
Collaborator Contribution Our collaborators have generated a platform for using NMR parameters in calculations of restrained molecular dynamics simulations. Using our NMR data, the first structural ensemble of a ribosome-nascent chain complex has been produced.
Impact A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding. Cabrita LD, Cassaignau AM, Launay HM, Waudby CA, Wlodarski T, Camilloni C, Karyadi ME, Robertson AL, Wang X, Wentink AS, Goodsell LS, Woolhead CA, Vendruscolo M, Dobson CM, Christodoulou J. Nat Struct Mol Biol. 2016 Feb 29. doi: 10.1038/nsmb.3182.
 
Description PEGylation of ribosome-nascent chain complexes 
Organisation University of Glasgow
Department College of Medical, Veterinary and Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have designed and generated a recombinant in vivo system using E.coli to produce ribosome-nascent chain complex system for the study of co-translational protein folding using NMR spectroscopy.
Collaborator Contribution Our collaborators have contributed an in vitro, coupled transcription-translation system for the producing of ribosome-nascent chain complexes, and PEGylation, a biochemical assay that has been used within our co-translational protein folding studies.
Impact A publication: A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding. Cabrita LD, Cassaignau AM, Launay HM, Waudby CA, Wlodarski T, Camilloni C, Karyadi ME, Robertson AL, Wang X, Wentink AS, Goodsell LS, Woolhead CA, Vendruscolo M, Dobson CM, Christodoulou J. Nat Struct Mol Biol. 2016 Feb 29. doi: 10.1038/nsmb.3182.
Start Year 2013
 
Description Outreach programme to A-level STEM students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact This outreach programme was designed for A-level pupils studying STEM based subjects and considering university options, though coming from schools whose students do not typically consider further education. A group of around 50 students participated in a range of activities hosted by the programme, including discussions with active researchers about their science.
Year(s) Of Engagement Activity 2015