| Transient complexes between proteins are essential in nature to ensure the proper functioning of signal-transduction and electron-transfer chains. Within such chains, a series of proteins work in relay to transfer electrons or to transmit signals between spatially distant areas of the cell. Proteins involved in these chains have to be able to recognize each other, perform the reaction and dissociate within the short time available for this reaction in the overall process. The emerging model of transient protein-protein interactions, based on solution NMR using paramagnetic labels, suggests the following: A dynamic encounter complex, in which the partners are loosely bound and sample a large range of conformations, coexists with the productive, single-conformation state from which the reaction occurs (productive complex). The dynamic encounter complex is less conformationally restricted than the productive complex thus facilitating the initial encounter. It provides a more efficient way to find the proper binding site by two-, rather than three-dimensional diffusion, and provides extra time for eventually needed conformational changes required for the reaction. How the dynamic encounter complex performs its role in transient protein interactions is still a mystery. In order to shed light on this question, we will investigate in molecular detail the structure and dynamics of the encounter state and its interplay with the productive complex. We will determine - The population of productive and dynamic encounter complex and - The conformational space sampled by the proteins in the dynamic encounter complex and the timescale of interconversion between different dynamic conformations. We follow a new approach that combines electron paramagnetic resonance (EPR) and fluorescence techniques (e.g. fluorescence correlation spectroscopy, FCS). Both partners of the complex will be spin labeled, such that the distance between the spin labels reflects the relative arrangement of the partners. Judicious choice of labeling positions will allow us to discriminate the productive and the dynamic encounter complex by distance. The distance between the spin labels will be determined from their dipolar interaction measured in liquid solution at room temperature by continuous wave (cw), high-field (95 GHz) and ultrahigh-field (275 GHz) EPR. From the full distance information (shape of the distance distribution, population), the conformational space of the proteins in the dynamic complex, the rate of transition between conformations and the relative populations of productive and dynamic complex will be obtained. The overall lifetime of the complexes will be determined by FCS and fluorescence cross correlation (FCC), making use of the change in diffusion time of the complex relative to the diffusion time of the constituent proteins. Combining the populations with the overall lifetime of the complex will yield the lifetimes of the productive and the dynamic encounter complex. We will study the following electron-transfer complexes: cytochrome c peroxidase:cytochrome c (Ccp:Cc) cytochrome f:plastocyanine (Cytf:PC). We have already investigated the properties of spin labels in these proteins and their complexes. They particularly qualify for the proposed project because biochemical studies and NMR investigations have already been performed on the spin labeled variants of these complexes. The project will be conducted in close collaboration with the group of Marcellus Ubbink in the Leiden Institute of Chemistry, which focuses on transient protein complexes, and the MicroSpectroscopy Centre at Wageningen University (head: Herbert van Amerongen), where extensive know-how and state-of-the-art instrumentation for fluorescence methods are available. |
