| When two proteins associate, first an encounter state is formed in which the partners are loosely bound. It has been proposed that this state speeds up the formation of the final, active complex; by diffusing over surface of the partner it is easier to form the active complex than via random collisions. Recent work from our group and others has shown that in weak protein complexes, the encounter state can represent a significant fraction of the total complex. Last year, an experimental method was proposed to characterize the elusive encounter state. Stabile radicals attached at various positions on the protein can be used as reporters to map the surface area visited by the partner protein during the encounter state. It has become clear that the encounter state cannot be represented by a single orientation, but rather consists of an ensemble. Until now it has not been possible to visualize this ensemble, because the technique to study it (paramagnetic NMR) provides only an average picture of all the protein orientations in the ensemble. The aims of the proposed research are to visualize the encounter state ensemble and determine which molecular factors influence it. With Brownian dynamics (BD) an ensemble that represents the encounter state will be created and this will be used to back-predict the expected NMR data. By comparison with the experimental NMR data, the model will be optimized. The rationale behind this approach is that the encounter state is most likely dominated by electrostatic interactions. This is the most long-range of interactions and NMR data indicate that within the encounter complex short range interactions are insignificant. BD programmes are suited for this application because they are designed to generate ensembles by repetitive docking of two proteins based solely on electrostatic interactions. The encounter state of the photosynthetic complex of cytochrome f and plastocyanin will be investigated. The complex from two cyanobacteria, Nostoc and Phormidium, will be studied. The Nostoc complex is known to be well-defined and dominated by charge interactions, while the Phormidium complex is weaker, more dynamic and less dependent on charges. The comparison will yield valuable data on what determines the degree of dynamics within the complex. The ensemble will be studied as a function of ionic strength and mutagenesis of plastocyanin surface residues. It has been proposed that at very low ionic strength protein complexes get ?frozen? due to very strong electrostatic interactions. This hypothesis can now be tested experimentally. Several surface residues on Nostoc plastocyanin appear to be dominant in determining the electrostatic interactions with cytochrome f. Site-directed mutagenesis will be used to establish their function for the encounter state. Formation of the encounter state is an important step towards the final, active protein complex, in particular in weak complexes. This study will yield a much better understanding of the factors that determine the nature of this dynamic state and will enable us to comprehend how the affinity between two proteins is tuned to match its biological function. |
