Analysis of the mechanism of protein secretion through the Sec machinery and exploitation as a polypeptide sequencing device

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Transport of proteins across membranes is a fundamental biological process essential for protein secretion andorganelle biogenesis1,2. This CASE project concerns the bacterial system, wherein protein transport from the cytosolacross the inner-membrane is usually achieved when the SecYEG protein-channel complex engages the cytosolic motorATPase SecA (secretion). Great strides have been made towards understanding the mechanism of protein-translocation:firstly, through the determination of the structures of the protein-channel and the motor components 3,4, and secondly,through the development of accurate and high-resolution assays for protein transport. We have developed such anassay, based on a split luciferase system, for both mitochondrial import and bacterial secretion5,6. The project willcontinue with the exploitation of this technology, together with a wide range of biochemical and biophysicalapproaches, towards the determination of the underlying molecular basis for protein transport. In parallel, your CASE project will be partnered with Oxford Nanopore Technologies (ONT) to explore the prospect ofexploiting the channel as a polypeptide sequencer. This would be achieved in the spirit of ONT technology developed forDNA sequencing - by monitoring variable conductance as different nucleotides of a single polymer pass through a porein the membrane. Currently, peptide sequencing is very challenging, time consuming and expensive, so if this can besimplified, and adapted for biological samples then the implications for analytical biochemistry and cell biology research,diagnostics, forensics etc. would be game changing. Early indications are very promising as we know that positivecharges (lysines and particularly arginines) and bulky residues struggle to make it through the channel and slowtransport considerably5. Therefore, different residues must have distinct interactions with the SecY-channel and mightalso elicit a measurable difference in conductance, and thereby generate interpretable signatures for different residuesrequired for sequencing. ONT is a globally successful company with a portfolio of technological innovations. Therefore,the partnership will bring together expertise of the Sec machinery together with sequencing know-how and platforms tocreate an ideal environment for the success of your project, for training and for experience of academic and biotechenvironments and teamwork.