Probing the energy landscape of conformational changes in membrane proteins

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The function of membrane proteins is often related to a structural rearrangement. These may be described by kinetic models that provide macroscopic transition rates for the transition from one conformation to another. However, these rates are mainly determined by the probability that a certain event occurs, not by the movement itself. The information about interactions that form and break during the movement is hidden in ensemble measurements. In this research proposal, we aim to resolve this "fine structure" of the energy landscape for structural rearrangements, oligomerization and protein folding using fluorescence spectroscopy from planar lipid bilayers. This information is in general not accessible due to the lack of synchronization between events. Following a stimulus, each event has a certain probability to occur. The start times are thus exponentially distributed over several milliseconds. The actual events are likely much faster (1ns-1us). We will circumvent this problem by studying single molecule fluorescence or measuring a parameter that observes the transition, not the start and final state. We will thus investigate (1) the protein translocation through an anthrax pore, giving insight into single amino acid interactions and protein folding; (2) protein oligomerization in planar lipid bilayer; and (3) resolve the speed of translocations and determine rotational movements in complex membrane proteins. With x-ray crystallography, we already have atomic scale images of proteins including membrane proteins. Nevertheless, in order to be able to securely manipulate their function with new drugs for inherited diseases or bacterial toxins with less side effects, we need to understand the movement of the proteins and their relation to the atomic scale models. The results will allow to assign directly single interactions between two amino acids to an event in the dynamics.