A biochemical and biophysical analysis of a ubiquitous protein translocation apparatus

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 & aggregate 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 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. Once prepared, various parts will then be labelled with chemical reagents that are able to report on their environment by the way that they interact with light. These probes will characterise the movements that the components make as they transport proteins across the membrane, which will aid our understanding of this reaction. New findings in this area will have implications in the understanding of protein secretion, cell biogenesis and development. Moreover, they will also help us understand other molecular machines and more general processes such as membrane transport. This information might be also exploited toward the development novel antibiotics.

Secretory and membrane proteins have to cross or be inserted into a phospholipid bilayer. This is achieved by translocation 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, which drives proteins through the channel. This proposal seeks to understand how this process works. Structures of SecYEG and SecA in their resting states have helped to describe how the passage of proteins through the membrane may occur. However, the structure of the open state or the active complex of the two has yet to be determined at high resolution. The protein path is located in the centre of the SecYEG monomer and a domain termed the 'plug' resides there to stabilise the closed state of the channel. Channel regulation must involve large conformational changes, including an opening of the protein pore and a displacement of the 'plug' from its blocking position. Experiments have been designed to address the timing, causation and nature of these conformational changes, occurring as substrate, channel and motor proteins combine to drive protein translocation. These experiments will rely on the incorporation of unique cysteines into the SecY complex, substrate polypeptide and a peptide mimic of the 'plug' domain. These strategically positioned sulphydryls will be coupled to various probes designed to sample and characterise their immediate environment. Measurement of various fluorescence parameters during translocation will be used to determine the nature of the probe's surroundings and its distance to other fluorescent object. In this way the movement of various domains of the apparatus and the substrate can be monitored. These findings will then be considered in view of the structure of the resting state to help us to understand the dynamics and mechanism of the reaction. They may also direct future efforts to selectively inhibit this essential reaction in bacteria.