Analysis of the of the interaction between the SecY protein translocation complex and its substrate pre-protein

Lead Research Organisation: University of Bristol
Department Name: Biochemistry


An ancient and essential development for life on Earth has been the evolution of a thin film of lipids that surround and form each cell. These membranes provide a barrier to water and hydrophilic solutes. They serve to isolate biological reactions from the outside, and offer the potential to separate charge and to communicate and combine with one another to form complex structures. More complicated cells also contain internal membrane structures that provide a further division for chemical reactions and generation of electric potentials. These events facilitated the ability to harness energy and to develop and maintain the complex structures and biochemistry of the cell. The necessary exchange of materials across lipid membranes between the outside and different compartments gives rise to a transport problem for small and large molecules alike. Proteins are large polymers of amino acids made according to the genetic code of each respective gene in the cell cytosol. In order to perform their specific roles many of them need to be specifically targeted and delivered to alternative locations. This requires that they pass either across or into a specific membrane. This proposal aims to learn more about this important process using the bacterial cell membrane as a model system. The apparatus responsible for protein movement across membranes has been purified. The interaction of this protein channel and its polypeptide substrate will be studies using a variety of biochemical and biophysical approaches that we have at our disposal. New findings in this area will have implications in the understanding of protein translocation. Moreover, they will also help us understand numerous other important problems in biology that involve the interaction of protein complexes with polypeptides.

Technical Summary

Secretory and membrane proteins harbour signal sequences to facilitate targeting to specific membranes where they are directed either through or into the bilayer. This is achieved by translocation of unfolded pre-proteins through the SecY/Sec61 complex, found in the ER membrane of eukaryotes or the plasma membrane of bacteria. The bacterial complex, SecYEG, associates with an ATPase SecA or ribosomes, which provide the thermodynamic drive to thread proteins through the channel by respective post- and co-translational mechanisms. This proposal seeks to understand how this process works. Two structures of SecYEG, one its membrane bound form and one in detergent solution have helped to describe how the passage of proteins through the membrane occurs. However, the structure of the open state of the protein channel has yet to be determined at high resolution. The protein path is located in the middle of the SecYEG monomer and maintained in a closed state by a central short 'plug' and a ring of hydrophobic residues. Adjacent to the channel is the signal sequence-binding pocket. Channel opening and the accommodation of the substrate polypeptide must involve large conformational changes in both these regions of the complex. Information on these events will be obtained by an analysis of the interaction of the substrate polypeptide with the bacterial SecY complex. Various strategies will be employed and include: fluorescence spectroscopy, chemical cross-linking, NMR and electron cryo-microscopy. In this way the nature and consequences of the interaction between the channel and its substrate will be characterised, to provide a picture of the active and open state of the channel. The results will also be relevant to our understanding of other numerous and important systems that involve non-covalent interactions of protein complexes with their peptide substrates and ligands.
Description See submitted grant report
Exploitation Route See submitted grant report
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

Description Wellcome Trust Senior Investigator Scheme
Amount £1,580,000 (GBP)
Funding ID 104632 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2014 
End 01/2020
Description The Bacterial Sec Machinery with Dr Andrew Woodland 
Organisation University of Dundee
Department Drug Discovery Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of samples and expertise for measurement of ATP driven protein transport through the bacterial Sec machinery
Collaborator Contribution Expertise for high-throughput analysis and exposure to large small chemical libraries to search fro potent inhibitors (potential anti-bacterial)
Impact Work in progress
Start Year 2012