Decoding the role of entropy in protein structural stability, dynamics, and function in health and disease - Proteins are critical to human health and defects in them produce innumerable diseases. How thermodynamics is manifested, at a molecular level, in the various properties of proteins is central to maximizing our understanding of biology and deriving an ability to intervene and resolve human disease. Yet, despite amazing progress in biochemistry, biophysics and structural biology, the nature and role of entropy in establishing protein structure-function relationships remains remarkably obscure. Incomplete knowledge of protein entropy, a basic component of the Gibbs free energy that controls most aspects of molecular biology, is an unacceptable deficiency. Our long-term goal is to remedy that deficiency. Here we focus on the nature and role of protein conformational entropy (DSconf) in the thermodynamics of protein function. In a long-term effort, we have developed robust NMR-based strategies to probe DSconf. We have discovered an unanticipated role for DSconf in molecular recognition by proteins. With the necessary tools now in hand and having revealed this foundation, we propose to determine the role of DSconf in critical biochemical arenas: allosteric regulation; membrane protein stability and function; and the enzyme catalytic transition state. One pathway to disease comes from dysfunction of allosteric regulation of proteins. Allostery remains incompletely understood. Though the classical treatments have evolved to an ensemble-based view, the molecular details of the thermodynamics underlying allosteric transitions are unacceptably incomplete. Our hypothesis is that many instances of allosteric regulation are critically influenced by DSconf. As a model system, we will dissect the underpinnings of allostery in the E3 ubiquitin ligase Parkin, mutants of which cause early onset Parkinson's disease. Our work will open new views and provide a basis for intervention. These studies will also promote investigation of other E3 ubiquitin ligases, defects of which give rise to a large array of diseases. What we know from NMR-based studies of protein internal motion, and the DSconf that motion represents, has largely been derived from soluble proteins. We have recently overcome potent barriers to applying our approach to integral membrane proteins (IMPs). The first examples strongly indicate that IMPs are dynamically distinct from their water-soluble counterparts. This raises profound questions about how conformational entropy is accommodated and used in IMPs. We will gain unprecedented insight by examination of a set of well-positioned IMPs with important biological functions such as ion transport, signaling and enzymatic activity. Finally, it has long been a mystery about how the bulk of the protein supports the transition state during enzyme catalysis. Using the serine proteases as a test bed, we will challenge the hypothesis that changes in conformational entropy influence the energetics of transiting from the ground state Michaelis complex to the transition state. In summary, this proposal is unified by a focus on conformational entropy and will advance our understanding of the thermodynamic underpinning of protein function in a range of biomedically important contexts.
