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

蛋白质的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学过程1，2。这个案例项目涉及细菌系统，其中蛋白质从胞浆中跨内膜运输通常是当SecYEG蛋白-通道复合体与胞液运动ATPase SecA(分泌)相结合时实现的。对蛋白质转运机制的理解已经取得了长足的进步：首先是通过确定蛋白质通道和运动成分3，4的结构，其次是通过发展准确和高分辨率的蛋白质运输分析方法。我们已经开发了这种基于分裂荧光素酶系统的分析方法，用于线粒体输入和细菌分泌5，6。该项目将继续开发这项技术，以及广泛的生化和生物物理方法，以确定蛋白质运输的潜在分子基础。同时，您的CASE项目将与牛津纳米孔技术公司(ONT)合作，探索将该通道用作多肽测序仪的前景。这将按照为DNA测序开发的ONT技术的精神来实现--通过监测单个聚合物的不同核苷酸通过膜中的孔时的可变电导。目前，多肽测序非常具有挑战性、耗时和昂贵，因此如果能够简化这一过程，并将其应用于生物样品，那么对分析生物化学和细胞生物学研究、诊断、法医学等方面的影响将是游戏规则的改变。早期的迹象是非常有希望的，因为我们知道正电荷(赖氨酸，特别是精氨酸)和大体积残留物很难通过通道，相当大程度上减缓了运输。因此，不同的残基必须与SecY通道有不同的相互作用，并可能引起可测量的电导差异，从而为测序所需的不同残基产生可解释的签名。ONT是一家全球成功的公司，拥有一系列技术创新。因此，合作伙伴关系将把SEC机制的专业知识与测序技术和平台结合在一起，为您的项目的成功、培训以及学术和生物技术环境和团队合作的经验创造一个理想的环境。

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.