Understanding the Mechanism of Membrane Protein Insertion

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All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. Cell membranes act as a molecular "skin", keeping the cell's insides in and separating different biochemical reactions. The barrier needs to be breached in a controlled manner to allow transport of nutrients, waste products and for communication with the outside world; this is achieved by a wide range of membrane-inserted proteins. We understand a great deal about the diverse biological functions that membrane proteins bestow, such as transport, respiration, photosynthesis. However, we know very little about how membranes are formed. In particular, the fundamental process through which proteins are inserted into membranes is poorly understood. Our proposal aims to address this outstanding problem. The process is facilitated by a number of different protein translocation systems (or translocons), including the ubiquitous Sec-machinery responsible for both protein secretion and membrane protein insertion. We aim to learn more about how this particular system works by studying an example from the common gut bacterium Escherichia coli. This is much more experimentally tractable than the human counterpart, but nonetheless should tell us a lot about how similar systems work in our own bodies. A collaborative project between the Collinson (Bristol) and Schaffitzel (Grenoble) Labs has for the first time succeeded in producing and assembling the complete bacterial membrane protein insertion machinery - aka the holo-translocon (HTL), composed of 7 individual subunits. The availability of this active machinery provides a unique opportunity to study the mechanism of membrane protein insertion. The molecular structure of the complex has been investigated, revealing a partially enclosed internal cavity that we have strong reasons to believe is composed of phospholipids. This lipid pool may provide a protected environment into which individual membrane-spanning segments of protein are inserted prior to their folding and release into the bilayer. This is an attractive hypothesis because it mirrors the way soluble (non-membrane) proteins are folded within a water-filled interior of large chaperone complexes.The proposal aims to build on these exciting developments to characterise the activity of HTL and explore the progression of an inserting membrane protein through the complex. An important first step will be to exploit our ability to reconstitute the insertion process from purified components and conduct a comprehensive analysis of basic biochemical rules and requirements of the machinery. The work will also employ new synthetic biology methods to overcome the limitations of the classical biochemical and biophysical approaches employed so far. Collinson and Jones (Cardiff) will combine forces to apply genetic reprogramming to introduce non-natural amino acids into proteins that allow the introduction of novel properties into target proteins. This technology will provide the tools to report on the environment of a protein during its passage into the membrane, as well as on the corresponding architecture of the HTL. Combined with the structure of the active complex, this information will challenge and develop the hypothesis involving the encapsulated insertion of membrane proteins. The results of the project will be important because they relate to an essential and fundamental biological concept, which may then lead to new ideas about its disruption for the development of anti-bacterial drugs. Moreover, the ideas and principles implemented and developed will be accessible to the analysis of other complex membrane protein systems.