Molecular Mechanisms of Sec-Mediated Protein Translocation

Living organisms are built of cells that are surrounded by and contain membranes consisting of lipids. Proteins are another important class of biological molecules. They are responsible for keeping all the biochemical processes running that a living cell needs to survive. Many of these proteins have to be transported through membranes, a process called translocation, or they have to be inserted into the membrane and, therefore, are referred to as membrane proteins. These tasks are fulfilled by particular membrane proteins, the translocons, which form special channels in the membranes. In the present project, we would like to understand the molecular mechanisms of the translocation process. In particular, we would like to know, how the channel decides, whether a given protein is transported through the membrane or inserted into it. Another problem is that many proteins, that have to be transported through the membrane, contain charged amino acids, thus, bearing charged groups. Such groups are normally repelled from the membrane, and the question arises of how the translocon deals with these cases. In the project, these questions will be tackled by computer modelling of the translocon located in the membrane of bacteria (called SecY). The structure of the bacterial channel will be taken from an open-access protein data bank. Then, special software packages will be used to put the translocon into a membrane and model movements of all atoms of the system, thereby mimicking the function of the channel. In addition, experimental studies of the translocation process will be performed by our collaborators. Since similar channels also exist in human cells, we expect important insights into the working principles of translocons that might become the basis for medical applications in the future.

About one third of all proteins are secreted across or integrated into biological membranes. One of the systems carrying out both the secretion (translocation) and the integration in bacteria is the SecYEG channel, also called translocon. It forms a pore in the membrane with an hourglass shape containing a constriction inside referred to as pore ring and a side exit into the membrane (lateral gate). The channel is sealed at the outside by a plug to prevent leakage of water and ions through the membrane in the resting state. The bacterial translocon is of interest in a medical context for developing anti-bacterial drugs and as a model for corresponding channels in the human body. In our project, we studied SecYEG with molecular dynamics simulations and electrostatic computations to learn about the physical mechanisms of the channels operation. Key results are: (i) Hydrophobic forces, which are responsible for protein folding in general, are also relevant for sealing the channel by binding the plug to the pore ring. (ii) To open the channel for translocation - in particular to move the plug out of the way - those forces have to be overcome. This is achieved by binding the signal sequence of the translocating protein (a kind of address label) to the lateral gate and by rearranging certain structural elements of the channel - called -helices - that traverse the membrane. The rearrangement is initiated by the reversible binding of a translocation partner - the SecA protein - to SecYEG, whereby energy in the form of the cellular energy currency ATP is consumed, and results in an opening of the plug. (iii) The electrochemical potential across the membrane has an influence on the helix arrangement and can principally keep the channel open in the absence of SecA. We hypothesize that this effect is responsible for the ion leakage observed in response to a change of the potential in experiments. (iv) Charged groups in the translocating protein are likely discharged as they move deeper into the channel. To optimize the electrostatic computations, we analyzed the dielectric properties of a model protein and other substances by employing a mathematical transformation of spectroscopic data known in physics as the Kramers-Kronig relations. Byproducts of this research are a mathematically sound implementation of the Kramers-Kronig relations and their application to analyze the physical properties of implanted antennas that are of relevance in medical diagnostics. Another byproduct of the project was fundamental theoretical research in the context of the aggregation of detergent molecules into micelles, which like the plug opening of SecYEG is a process depending on hydrophobic forces.