Transient protein complexes: structures and dynamics

Short-lived protein complexes represent a new area of biochemical research. These complexes are formed whenever a high turn-over of the complex is required for its biological function, for example in a chain of electron transfer proteins or a cascade of reactions. The high dissociation rate makes binding weak in these complexes, and thus there is a need for a compromise between turn-over and specificity. The aim of the research is to understand at atomic level how these two factors are controlled. So far my research has demonstrated that there is a surprising variety in the nature of transient complexes. In some complexes, the partners assume a single-orientation. In others, the partners are in a dynamic ensemble of many orientations, thus lacking specificity in binding. I have determined the structures of two single-orientation transient complexes. The three key objectives of the work comprise the following. 1) Characterisation of dynamic ensembles using NMR relaxation measurements. 15N relaxation measurements on both proteins in a dynamic complex will be used to probe their rotational freedom. Also protection from soluble paramagnetic relaxation agents will be analysed. 2) Determination of structures of transient complexes. To understand what properties (electrostatics, hydrophobics, H-bonding) play a role in the interactions, structures of transient complexes need to be obtained. For this purpose, new, paramagnetic NMR approaches are developed. 3) Study of transient complexes in environments that are similar to cellular conditions, to understand the effects of molecular crowding on complex formation. The effects of crowding agents on very weakly bound transient complexes will be analysed. The research will lead to a comprehensive picture of the transient complexes and will contribute to a general understanding of the factors that determine specificity of macromolecular binding.