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

Transport of proteins across membranes is a fundamental biological process essential for protein secretion and
organelle biogenesis1,2. This CASE project concerns the bacterial system, wherein protein transport from the cytosol
across the inner-membrane is usually achieved when the SecYEG protein-channel complex engages the cytosolic motor
ATPase 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 an
assay, based on a split luciferase system, for both mitochondrial import and bacterial secretion5,6. The project will
continue with the exploitation of this technology, together with a wide range of biochemical and biophysical
approaches, 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 of
exploiting the channel as a polypeptide sequencer. This would be achieved in the spirit of ONT technology developed for
DNA sequencing - by monitoring variable conductance as different nucleotides of a single polymer pass through a pore
in the membrane. Currently, peptide sequencing is very challenging, time consuming and expensive, so if this can be
simplified, 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 positive
charges (lysines and particularly arginines) and bulky residues struggle to make it through the channel and slow
transport considerably5. Therefore, different residues must have distinct interactions with the SecY-channel and might
also elicit a measurable difference in conductance, and thereby generate interpretable signatures for different residues
required 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 to
create an ideal environment for the success of your project, for training and for experience of academic and biotech
environments and teamwork.