Ensemble and single molecule analysis of protein translocation

All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. These act as an ideal 'skin', keeping the cell's insides in! In the absence of other components they would act as barriers, preventing the necessary rapid exchange of nutrients and waste products, and of larger molecules like proteins, between the environment and the cell interior. Such passage is required for many proteins to perform their biological functions - for example the abundant protein albumin of the blood has to be secreted across the membrane from its site of synthesis in liver cells. To overcome this potential problem, biological membranes contain a number of translocation systems that enable proteins and other useful substances ('substrates') to pass across the phospholipid barrier. In the case of protein substrates, these translocation systems recognise the specific proteins to be translocated via signals embedded in the sequence of amino acids from which they are constructed. We aim to learn more about how such translocation systems work by studying an example from the common gut bacterium Escherichia coli, which is experimentally easier to work with than human cells, but nonetheless should tell us a lot about how similar systems work in our own bodies. Like our own, the bacterial translocation system (the 'translocon') serves to secrete proteins from the interior of the cell to the outside. It comprises two components - a three-protein complex named SecYEG that forms a channel through the membrane, and a motor protein named SecA that drives the passage of proteins through the channel, fuelled by energy provided by ATP, the so-called 'energy currency' of the cell. We know that the energy for protein translocation is released when the motor protein SecA breaks down ATP into two smaller molecules, ADP and phosphate. What we don't understand is how this process actually drives movement of the translocating protein. However, it is clear that a cycle of changes in the shapes of SecA and SecYEG, termed conformational changes, are likely to be involved, much as the movements of pistons and cams are involved in internal combustion engines. It is these conformational changes that will be explored in the proposed project. To do this, we will use recombinant DNA techniques to introduce the amino acid cysteine into the protein substrate and at places in the translocon that we suspect move during the translocation process. This particular type of amino acid is chemically reactive, meaning that we can selectively attach fluorescent or magnetic probes with which we can monitor the environment at each place during different stages of protein translocation and ATP breakdown. In particular, the distances between pairs of probes can be measured by physical techniques known as Förster resonance energy transfer (FRET) and electron spin resonance (ESR) respectively. We will also examine whether pairs of cysteines are sufficiently close to each other to be chemically linked together by cross-linking molecules of defined length, and if so, we will see what effect this tethering together has on the function of the translocation machinery. These types of experiments, conducted in the test tube on millions of translocons at a time under so-called 'ensemble' conditions, should be very revealing of the mechanism. However, in such ensembles it is very difficult to synchronise the translocation 'machines' so that they are all simultaneously at the same stage of their mechanical cycles when we observe them. To complement this approach we will therefore also take advantage of the development of very sensitive microscopy techniques, which will allow us to follow the conformational changes of a single translocon, and the associated translocation of protein, at a time. Taken together, the ensemble and single molecule approaches should allow us to understand the inner workings of a molecular machine essential in all cells.

Protein secretion in bacteria utilises SecA to drive protein through the ubiquitous SecYEG complex. In spite of our knowledge of the structure, and of the stages and timing of the ATP hydrolytic cycle, we understand little about the corresponding conformational changes. We therefore propose a multi-disciplinary programme to explore the dynamics of the translocation machinery. To this end, cysteines will be incorporated to enable selective modification at specific sites in SecA, SecYEG and pre-protein substrate. Intra- and inter-molecular cross-links between specific thiol pairs in SecA and SecYEG will be used to monitor their relative locations in the presence of ADP, ATP (AMPPNP), and when engaged in translocation. Fully cross-linked samples will also be characterised with respect to ATP hydrolysis and pre-protein transport. In addition, the introduction of single or pairs of fluorescent or paramagnetic probes will be used to report on their environment and spatial relationships (e.g. distance and orientation). FRET will be used to monitor nucleotide- and pre-protein dependent conformational changes within SecA and SecYEG. In parallel, ESR spectroscopy will be employed to provide reliable distance constraints between given points of the complex at different stages of the translocation cycle. Ensemble experiments will be complemented by investigations at the single molecule level, using total internal reflection fluorescence microscopy (TIRF). These will allow us to circumvent problems associated with inherently inefficient ensemble assays of transport, and the difficulty of synchronising populations of translocating complexes. It should thereby be possible to follow translocation in real time, including conformational changes in SecYEG and SecA. Taken together, these approaches should contribute greatly to an understanding of the molecular mechanism of protein translocation, a process of critical importance to all cells.

The main objective of the proposal is toward the understanding of an essential and ubiquitous reaction in cells: protein translocation across membranes. Therefore, the immediate impacts will be in the scientific advancement in the areas of membrane biology, molecular motors, protein dynamics as well as the development of novel biophysical techniques to observe such systems. In addition, it will also offer training in multi-disciplinary research to its post-doctoral workers, which will equip them with new skills and give them essential experience for research or related jobs in academia, education, healthcare, or industry. In addition to the project itself, we anticipate considerable added benefit and impact through collaborative links already established nationally and internationally, particularly amongst other academic institutions in addition to Bristol, Leeds and Oxford. New findings will be disseminated both through peer-reviewed publications and by presentations at international scientific meetings. The output resulting from this project will also be relayed to the media via press release and high profile sites on the Universities' front web pages. Moreover, we will continue our commitment and track-record in public engagement. This involves communication of the excitement of science to the both junior and adult members of our society. The immediate beneficiaries of this project will clearly be the wider UK and international academic communities, public and private education, the healthcare sector and industry. However, in the medium term we foresee potential impacts in the areas of antimicrobial drugs and in nanotechnology. The former reflects the fact that while the translocon per se is ubiquitous, the motor protein SecA, is found only in bacteria. Given the growing problem of antibiotic resistance in the UK and worldwide, such development would have a huge impact on human and animal health. Our findings on the conformations and dynamics of this protein will open the way to the development of novel antimicrobial drugs by the pharmaceutical industry. And in this endeavour we propose to act positively. Should the grant be funded we will set in motion the initial applications for seed-funding and the installation of experimental approaches to inhibitor design of this bacteria specific process. In doing so we will enlist the existing entrepreneurial infra-structures in Bristol and Leeds. The second potential biotechnological application relates to the aspect of the proposal that seeks the understanding of molecular machines utilising chemical energy for molecular motion of proteins across the barrier of the membrane. Activities such as these are of course potentially extremely useful to us. Moreover, our greater understanding of them, particularly in the dynamic energy coupling steps, will enhance our ability to manipulate them accordingly. This could indeed prove useful in the development of novel activities pertinent to bio-sensation or the construction of nano-machines or motors, relevant to the BBSRCs commitment to synthetic biology. Therefore, as the opportunity arises these anticipated spin-offs will be exploited to the full.