A complete description of biomolecular activity requires an understanding of the nature and the role of protein conformational dynamics. In recent years novel nuclear magnetic resonance (NMR) techniques have emerged that provide hitherto inaccessible detail concerning biomolecular motions occurring on physiologically important timescales. In particular residual dipolar couplings (RDCs) provide precise information about time and ensemble averaged structural and dynamic processes with correlations times up to the millisecond, and thereby encode key information for understanding biological activity. In recent years we have developed two very different approaches to the quantitative description of intrinsic protein motions on a wide range of timescales using RDCs. Application of these techniques to the study of the proteins Ubiquitin (Ub) and GB3 resulted in the convergent observation of enhanced dynamic fluctuations occurring on intermediate timescales (nano to millisecond) in the physiological interaction sites of these proteins. The motions occurring in these interaction sites were suggested to exhibit specific modes that would either optimally accommodate the interaction partner, or to intrinsically sample the conformations found in complex with diverse functional partners. In this study we will investigate, for the first time, the nature and timescale of these slower motions, not only in the isolated proteins, but also in the presence of different interaction partners. Concentrating on a specific and important cellular paradigm, we focus on characterizing the interaction between Ub and different Ub binding domains (UBDs). Ub is a versatile cellular signal, regulating a wide variety of activities ranging from protein degradation and quality control, endocytosis, transcriptional regulation to cell signaling and membrane trafficking. Ub has a large number of intracellular partners (more than 150 have been annotated), and while affinities of mono-Ub-binding interactions are very often weak, they span two orders of magnitude (Kd 3-2000µM). We will study the interaction between Ub and a range of UBDs with different affinities in order to probe the possible link between the structural dynamics of the molecular complex occurring on timescales up to the millisecond, and the kinetics of the interaction. In order to achieve this goal we will build on and extend state-of-the-art experimental, analytical, numerical and molecular simulation techniques established over the last ten years in the laboratory of the coordinator to measure and analyse RDCs, spin relaxation and relaxation dispersion from diverse Ub-UBD complexes. During the course of this project we will (a) compare, for the first time, the nature of large scale slow motions in the presence and absence of functional partners, (b) establish the dependence of interaction affinity on molecular recognition dynamics in free and bound forms of interacting partners, (c) extend the timescale over which NMR can be used to determine local entropic and enthalpic contributions to the thermodynamic equilibrium (d) more than double the number of individual proteins whose backbone dynamics have been characterised using RDCs, thereby establishing general trends concerning the nature of slow motions across different protein families. In summary, this project fully exploits the unique sensitivity of NMR to study weak protein-protein interactions at atomic resolution to address a problem of great current importance. While methods have been developed to describe slower motions in proteins, with the observation that these dynamics tend to occur in the interaction site of the proteins, the modulation of this flexibility upon interaction remains unknown. Methods developed in the group of the coordinator over the last decade will be applied in the course of this project to compare protein dynamics in the free and bound forms of Ub and UBDs participating in weak complexes.