Mechanisms and dynamics of allosteric function in proteins

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Abstract Biology is driven through the action of proteins. We know that structure often provides the foundation for proteins’ function, but in recent years it has become clear that protein function is also critically dependent on dynamics, or movements of structure. How dynamics enables function is now a central question in protein biology that limits our basic understanding of proteins, as well as applications in drug discovery and protein design. While there are many types of functions that dynamics – or conformational flexibility – promotes, two functional archetypes for dynamics are enzyme catalysis and allostery. The mechanistic bases for these two phenomena, pervasive as they are, remain largely mysterious and have attracted much attention for the likely role of dynamics. The Lee laboratory has focused on studying dynamics and allostery in proteins using NMR and other biophysical methods for nearly 20 years. The approach outlined in this proposal is to combine investigation of natural allosteric enzymes (Areas 1 and 2) with efforts to engineer allosteric regulation into signaling proteins using optogenetics (Area 3). In the last five years, the lab has developed two complementary systems for NMR and biophysical studies of dynamics and allostery that are highly amenable for addressing these mechanistic questions and, importantly, developing approaches to study intersubunit allosteric communication. The two systems are the enzymes chorismate mutase (CM) and thymidylate synthase (TS), both symmetric homodimers that are functionally allosteric. CM (from yeast) is a classically allosteric protein, exhibiting all the hallmarks of traditional allostery: sigmoidal activity curve; symmetric quaternary structure; tense (“T”) and relaxed (“R”) conformations; and small molecule allosteric effector ligands that either up- or down-regulate activity. In contrast to CM’s positive cooperativity, TS is negatively cooperative because it is half-the-sites reactive. Work will be on the E. coli (ecTS) and human (hTS) forms, which, despite their similarities show very different behaviors. The human TS is the target of anticancer drug 5-fluoro-uracil (5-FU). CM, ecTS, and hTS all have outstanding features for study by solution NMR since they are highly soluble, stable, and yield excellent spectra. The goals for the next five years fall into three main areas: (1) Through the use of NMR spectroscopy, molecular dynamics simulations, calorimetry, x-ray crystallography, and biochemistry, the structural and dynamic properties of these enzymes will be related to functional behaviors of key interest, such as: allosteric communication; how apo state conformations compare to T and R conformations; protomer asymmetry in singly liganded states; and the nature of the transition state. (2) We will advance the study of protein homodimers by NMR by introducing a technology for chemical conjugation of protomers using click chemistry. Mixed labeled dimers produced this way will facilitate NMR study of interprotomer interactions, such as allostery, and improve NMR structure determination of homodimers. (3) For engineered GTPases that have been artificially placed under optogenetic control, the allosteric mechanisms will be determined using an NMR approach.