Molecular Evolution and Evolution-guided Protein Engineering

Abstract

The theory of evolution unifies all aspects of biology. The forces of natural selection that drive allelic variation and the morphological diversification of populations and organisms are the same as those that compel biomolecules to acquire novel properties and functions. Indeed, the myriad of molecular processes that permit complex life - signaling, catalysis, and the regulation of cellular processes, among them - are the products of molecular refinement over innumerable generations of descent with modification since the emergence of the last universal common ancestor approximately 4.6 billion years ago. Studying the mechanisms of molecular evolution not only provides retrospective insight into the processes that have shaped modern biology, but also profound insight on the fundament physical and chemical properties of macromolecular biomolecules (particularly proteins and DNA). Moreso, understanding how proteins evolve phenotypes when challenged by a changing environment can provide great utility in protein engineering endeavours; knowledge of how proteins acquire novel functions in nature can guide the strategies that protein engineers use to design new-to-nature proteins, for example in biocatalysis.

The work presented within this thesis centers on studying the mechanisms and processes of protein molecular evolution. Through computational phylogenetic reconstruction, ancestral sequence reconstruction, and molecular simulations as well as experimental protein characterization, the following chapters each build on established elements, or present new aspects of molecular evolution within distinct protein folds and families. Each independent study is unified into a single body of work by the methods and overarching themes of molecular evolution that are broadly shared among all extant proteins, instead of by the protein systems under investigation themselves. The insights provided from these studies are discussed not only within the context of evolution, but also under the broader lens of the protein sequence-structure-function-dynamics paradigm, and often extended to discussions around applications within protein engineering and synthetic biology.

Chapters 1, 2 and 3 each provide introductions and literature reviews of the core concepts discussed within this thesis: the principles and technical perspectives of molecular phylogenetics and the methods therein, ancestral sequence reconstruction and its utility in protein engineering and the evolutionary and structural strategies in rational protein (biosensor) design, respectively. Chapters 4 and 5 each present a comprehensive phylogenetic analysis: In chapter 4, extensive phylogenetic analyses resolve long-standing questions within serine protease inhibitor (serpin) biology and evolution, particularly on the origin of the enigmatic prokaryotic serpins. Through phylogenetic analysis and complementary computational methods, as well as structural characterization, the work presented in chapter 5 identifies a novel clade of ribonucleotide reductases (RNRs) - an enzyme required by all DNA-based life - ancestral to modern RNR lineages, highlighting the mechanisms of RNR functional divergence and shining light on the biogeochemical trends that shaped their evolution. Chapter 6 presents an ancestral sequence reconstruction (ASR) study on the emergence and activity-stability trade-offs of the Ideonella sakaiensis PETase, a poly(ethylene) terephthalate degrading enzyme of great industrial biocatalytic interest. Chapter 7 presents a study that uses ASR, next-generation sequencing, molecular simulations, and in vitro binding assays to show that DNA recognition in LacI family transcriptional regulators is a highly metastable trait that has evolved on a rugged fitness landscape. Finally, chapter 8 concludes the work presented in this thesis with a discussion on the perspectives gained from each chapter and the directions that the field of molecular evolution is moving towards. Show more