Intellectual Merit: Proteins are molecular machines that carry out the work of cells, tissues and organs and ultimately orchestrate the development of individuals. In spite of the central role proteins play in the biology of life, the mechanisms promoting the evolution of protein functions remain unclear. Ideally, proteins of ancient organisms would be studied in order to understand how function changes over time and how the same function can evolve independently in distinct proteins. Because ancient organisms are extinct, this study will use a paleomolecular approach to resurrect extinct proteins to study the history of protein functional evolution. For this research, a 300 million year old ancestral protein and its descendants will be synthesized for a group of plant metabolite-producing enzymes in order to test several hypotheses. The first hypothesis to be tested is that the ancestral enzymes begin as generalist "jack-of-all trades" and later give rise to specialized descendants after gene duplication. This hypothesis has never been tested for any diverse enzyme family and thus the results will fill a void in understanding the pattern of protein functional diversification. The second hypothesis to be tested is that numerous distinct mutational paths exist which allow different ancestral proteins to evolve similar functions. In this case, the mutational paths leading to the independent evolution of caffeine synthesis activity in the coffee and tea lineages will be recapitulated. Convergent evolution of enzyme activity has never been investigated at this level before so this study will illuminate the detailed mechanism by which it occurs.
Broader impacts: This study will provide insight into plant defense and the production of important secondary compounds like caffeine, thereby having potential implications for agriculture and metabolic engineering of plants. In addition, this research may have implications for protein engineering because ancestral enzymes provide useful points from which to initiate selection for desirable activities because of their broad spectrum of capabilities. Undergraduate and graduate students will perform all of the laboratory experiments required for this study and present their results at conferences and as manuscripts detailing their findings. Also, the conceptual issues presented here are implemented as one theme in a senior-level Molecular Phylogenetics and Evolution course in which adaptive hypotheses for protein evolution are proposed and statistical tests are designed to test the predictions. An evolutionary theme also forms the foundation of a companion Molecular Biology Laboratory course in which experiments are used to test hypotheses related to the effects of natural selection on protein functional evolution by performing gene cloning, protein expression, enzyme assays, and site-directed mutagenesis. As such, the proposed research and these two courses provide students with an integrated and conceptual perspective on protein evolution that unites their training in chemistry and biology.