Emergence of New Regulatory Mechanisms
Protein regulation is ubiquitous in biology. It plays an essential role in controlling and coordinating a variety of cellular processes including replication, division and growth. Protein mis-regulation has been implicated in many human diseases. The emergence of new regulatory strategies often requires protein conformational heterogeneity. For example, allostery requires the ability to toggle between two (or more) distinct polypeptide structures. Similarly, a protein’s ability to interact with multiple binding partners often requires sampling a range of conformations. Past experimental studies of individual polypeptides have yielded a molecular level understanding of many conformation-dependent regulatory processes. Despite this fact, we know little about how amino acid sequence changes serve to alter a protein’s conformational landscape, such that new regulatory strategies can be realized. The goal of this project is to illuminate how evolutionary trajectories, shaped by natural selection, provide access to new protein conformations, which facilitate the emergence of novel regulatory mechanisms.
Enzyme Recruitment and Metabolic Innovation
Environmental change drives the evolution of new metabolic pathways. A key step in metabolic innovation is enzyme recruitment — when an existing catalyst is enlisted to provide a new function. Enzyme recruitment is powered by the inherent functional promiscuity of modern enzymes, which allows them to transform multiple substrates and perform multiple chemical reactions. Indeed, recent estimates indicate that a single bacterial proteome likely harbors thousands of promiscuous enzymatic activities “lying in wait” for future recruitment. Such widespread promiscuity suggests a multitude of potential solutions to new metabolic challenges. Yet the solution selected by evolution remains unpredictable, in part, because enzyme recruitment occurs amidst a complex, dynamic physiological backdrop, and factors that facilitate or constrain the recruitment process remain unidentified. The goal of this project is to identify specific biological factors that contribute to enzyme recruitment outcomes by characterizing individual recruitment events using a combination of adaptive laboratory evolution, classical biochemistry and modern -omics methods.
Allosteric Regulation in Monomeric Enzymes
Human glucokinase catalyzes the ATP-dependent phosphorylation of glucose in the first reaction of glycolysis. This chemical transformation is the rate-limiting step of glucose metabolism in the human liver and pancreas. As such, glucokinase is a central regulator of glucose homeostasis. Genetic lesions within the glk gene that impair function result in maturity-onset diabetes of the young (MODY), while mutations that enhance catalysis cause hyperinsulinemia of infancy. These two disease states demonstrate the importance of precisely regulating glucokinase activity in vivo and emphasize the potential of therapeutically manipulating this key metabolic enzyme. Indeed, small-molecule glucokinase activators that enhance catalysis by as little as 6-fold are potential diabetes therapeutic agents. Glucokinase is a monomeric enzyme with the unique ability to be allosterically regulated by its substrate, D-glucose. The steady-state velocity of glucose phosphorylation is not hyperbolic, but instead displays a sigmoidal response to increasing sugar concentrations. The mechanistic basis for this kinetic cooperativity is unknown. Several models of kinetic cooperativity in monomeric enzymes have been formulated, yet none have been experimentally proven for human glucokinase. The goal of this project is to elucidate the mechanistic basis for cooperativity in human glucokinase. We wish to provide a kinetic and molecular description of the slow conformational transitions that are believed to give rise to cooperativity. We also wish to understand the mechanism of activation afforded by individual mutations in the glk gene that lead to hyperinsulinemia.