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

蛋白质跨膜运输是蛋白质分泌和细胞器生物发生所必需的基本生物过程1，2。该CASE项目涉及细菌系统，其中蛋白质从胞质溶胶穿过内膜的转运通常是当SecYEG蛋白通道复合物与胞质溶胶马达ATP酶SecA（分泌）接合时实现的。在理解蛋白质转运的机制方面取得了很大的进展：首先，通过确定蛋白质通道和马达组件的结构3，4，其次，通过开发精确和高分辨率的蛋白质转运测定。我们已经开发了这样的一个分析，基于分裂荧光素酶系统，为线粒体输入和细菌分泌5，6。该项目将继续开发这项技术，以及广泛的生物化学和生物制药方法，以确定蛋白质转运的潜在分子基础。与此同时，您的CASE项目将与牛津纳米孔技术公司（ONT）合作，探索利用通道作为多肽测序仪的前景。这将在ONT技术的精神下实现，该技术是为DNA测序而开发的--当单个聚合物的不同核苷酸通过膜上的一个孔时，通过监测可变的电导来实现。目前，肽测序非常具有挑战性，耗时且昂贵，因此如果可以简化并适用于生物样品，那么对分析生物化学和细胞生物学研究，诊断，法医学等的影响将改变游戏规则。早期的迹象是非常有希望的，因为我们知道，正电荷（赖氨酸，特别是赖氨酸）和庞大的残基难以通过通道和缓慢的运输。因此，不同的残基必须与SecY通道有不同的相互作用，并且可能也会引起电导的可测量差异，从而为测序所需的不同残基产生可解释的特征。ONT是一家全球成功的公司，拥有一系列技术创新。因此，该合作伙伴关系将把SEC机器的专业知识与测序技术和平台结合起来，为您的项目成功创造一个理想的环境，为学术和生物技术环境和团队合作提供培训和经验。

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.