Methods in protein engineering and screening : from rational design to directed evolution and beyond

This chapter is the culmination of several successful collaborations during my tenure as a graduate student. The main theme presented in this chapter is molecular modeling and how computational tools can be applied to the post facto rationalization of laboratory evolution experiments (Sections 1-3). Examples of the predictive power of modern computational approaches are also discussed. In section one, we find that mutations observed in Citrate Synthase (CS) during the Lenski long-term evolution experiment (LTEE) occurred both before and after Escherichia coli evolved to utilize citrate aerobically as a carbon source. Detailed kinetic, molecular modeling, and metabolic flux analyses provide support of a model of evolution where mutations in CS were needed to first potentiate and then later refine central metabolic regulatory networks for the use of the newly available nutrient. In section two, we addict a diverse set of bacteria to noncanonical amino acids through engineering TEM-1 β-lactamase. The creation of rotamer libraries and simulations provide mechanistic clues to the addiction of the enzyme to two tyrosine analogs and may provide a framework for future engineering efforts of other enzymes. Extending upon our noncanonical amino acid work, section three rationalizes rifampicin resistance in Escherichia coli in the context of standard and extended genetic codes. Finally, section four uses homology modeling, molecular dynamics simulations, and docking studies in an attempt to explain differences in thiabendazole (TBZ) resistance in human and fungal β-tubulins. While covering seemingly disparate topics, the training in the use of various software packages provided me with a diverse toolkit that would aid in my development as a scientist and taught me to appreciate structure-function relationships at a much deeper level. Lessons learned in these four sections would help shape projects described hereafter. 37 2.1 FINE-TUNING CITRATE SYNTHASE FLUX POTENTIATES AND REFINES METABOLIC INNOVATION IN THE LENSKI EVOLUTION EXPERIMENT Introduction Natural selection has provided the world with a vast array of diverse life forms, each with complex regulatory mechanisms and metabolic processes. Metabolism, life’s chief use of chemistry, has been brilliantly crafted by evolution to enable organisms the ability to occupy niches over a staggering range of environments. The ability to adapt to a changing world, encoded by rare genetic mutations, gives rise to varying levels of fitness, which then in turn, leads to expansion of inherited traits and observable phenotypes (Ryall et al., 2012). In nature, this process rarely happens on observable timescales (Barrick and Lenski, 2013). However, the Lenski long-term evolution experiment (LTEE) has provided key insights into how metabolic and regulatory networks have evolved in Escherichia coli under laboratory conditions and gives clues as to how evolution works in nature. The LTEE has been an ongoing endeavor for more than 25 years and continues to enrich our understanding of evolutionary forces and provides us with a dataset from which to design and test new hypotheses (Lenski and Travisano, 1994; Lenski et al., 1991). The ability of a single population to aerobically utilize citrate as a carbon source is perhaps the most widely regarded result of the LTEE to date. After roughly 31,500 generations (~15 years), only one of the original twelve populations evolved the ability to colonize this ecological niche, giving access to an abundant nutrient not previously accessible (Blount et al., 2008). After more than a decade since the observation of this phenotype, none of the other populations have gained this metabolic trait. Due to the rarity of this innovation, it is clear that a multi-step mutational pathway was necessary to occupy this new niche. Three stages of evolution were required in the LTEE for robust citrate utilization: potentiation, actualization, and refinement. The first appearance of Cit+ cells is referred to as the actualization step and was caused by a promoter duplication event that allowed the citrate:succinate