Evolutionary-based protein engineering of binding proteins and enzymes

Abstract

The work in this thesis applies evolution-guided protein engineering to understand and design protein function. Ancestral sequence reconstruction, biophysical and enzymatic characterisation, and structural analysis are used to reveal mechanisms of molecular evolution that shape specificity and catalysis and how these mechanisms can be used to uncover new functionality in engineered proteins and enzymes, highlighting the broad applicability of these methods across diverse systems.

Chapter 1 provides the conceptual framework for protein design and an overview of the key evolutionary methods used throughout the thesis.

Chapter 2 examines the evolution of ligand binding from a thermodynamic perspective, using thermodynamics as a language to describe changes in affinity, specificity, and binding energetics over evolutionary time. This chapter also serves as an extended introduction to the included research articles. The first research article presents a comprehensive characterisation of ancestrally reconstructed LacI/GalR family transcription factors and shows that changes in binding specificity along the evolutionary trajectory of Escherichia coli LacI are driven by enthalpy-entropy trade-offs in response to environmental pressures, including temperature. In the most distant ancestor, binding is entropically driven via entropic redistribution and retained flexibility, highlighting the role of protein dynamics in the evolution of ligand specificity. These concepts are further developed in the published review article included in this chapter.

Chapter 3 demonstrates how evolution-guided design can be used to engineer enzymes with novel catalytic functions, including biocatalysts for degradation and recycling of plastics, specifically polyethylene terephthalate (PET), nylon 6 and nylon 6,6. The first research article reconstructs evolutionary sequence space from PET-degrading cutinases to identify functional variants and reveal convergence among PET lineages. The second research article applies a similar strategy to evolve nylon 6,6 oligomer-degrading enzymes from serine-protease nylonases, discovering a novel class of nylon 6,6 hydrolases. Structural analysis shows this specificity shift arises from epistatic active-site mutations that enable favourable electrostatic interactions and improved complementarity of binding site size and shape.

Chapter 4 synthesises these findings into a general discussion of key ideas and considers future directions in protein engineering, including the growing role of computational protein design in linking protein genotype and phenotype.