Dynamic allostery of Sec machinery in protein transport and folding

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All cells are surrounded by oily membranes keeping the cell insides in, and unwanted substances and parasites out. Thus, selective pores have evolved for the supply of nutrients and removal of waste products. Without these 'transporters' the membrane presents an impermeable barrier, particularly for larger molecules like proteins. Such passage is required for many proteins to perform their biological functions, such as secretion of antibodies into the blood stream by immune cells. Therefore, biological membranes also contain a number of translocation systems that recognise the specific proteins to be translocated via signals embedded in the sequence of composite amino acids.We aim to learn more about how one such translocation system works in bacterium Escherichia coli: the "translocon", which secretes proteins from the interior across the membrane and also into it; essential even for basic survival. The machinery comprises two components -a protein-channel embedded within the membrane, and a motor protein named SecA that drives the passage of proteins through the channel, fuelled by energy provided by ATP, the so-called "energy-currency" of the cell.The energy for protein translocation is released when SecA breaks down ATP into two smaller molecules, ADP and phosphate and this process is coupled to the movement of the translocating protein in a cycle of changes to the shape of SecA and the channel, termed conformational changes. Since there are two partners, we are particularly interested how their movement is coordinated. Our objective is to understand how this "dance" is choreographed in time and space, i.e. take a molecular movie of the pair.We have developed tools, which allows us to interrogate one component at a time with a high temporal resolution by illuminating one of the partners at a time. This is achieved with rapidly alternating laser pulses, while recording their behaviour by aiming very sensitive and fast detectors (or cameras) onto the dance floor. To do so, we have to use genetic and biochemical techniques to introduce optical reporters (fluorescence probes) onto the proteins, in places that move during the translocation process, or dance (if you prefer our colourful metaphor!).These fluorescent probes report on the specific motions during different stages of protein translocation and during ATP breakdown. In the past few years, we have had considerable success in this approach observing molecular gyrations of individual partners. Surprisingly, we found the translocon dances about ten times faster than its energy providing motor partner. Now, we want to figure out how this is possible, and how such a strange dance is coordinated. To do so we will observe movies of both partners simultaneously, which requires considerable molecular biology craft for the introduction of the right reporters in the right places. We also need more elaborate and faster laser systems to illuminate the dance floor and of course fast rolling cameras and other detectors. After the movie is recorded, we will tease out useful information about the dance from individual movies that are encoded in the detected signals. To do this we have developed computational algorithms to convert the extremely rapid signals into meaningful information. The findings will uncover details of the conformational dance and explain fundamentally how the coordinated, and paradoxically incoherent, behaviour of the motor and channel result in the passage of proteins across and into the membranes of cells. This information could also inform us about how very different dancers move elsewhere in biology, even with different routines. So, we could learn more how ATP is also used to, help move different types of cargo around the cell, and for protein quality control. Moreover, this knowledge could be exploited in the development of compounds that target those elements (dancers!) that are unique to bacteria, potentially for highly desirable novel antibiotic.