The structure, dynamics, function and evolution of binding proteins

Abstract

Macromolecular recognition - the ability of biological molecules (especially proteins) to produce physiological responses by forming specific, high-affinity, non-covalent complexes with other molecules - underlies every biological process, including cellular signalling, immune recognition and enzyme catalysis. Determining the chemical and physical factors (including the structure and dynamics of the interacting molecules) that govern the properties of macromolecular recognition events is central to appreciating how biological molecules work and why disease occurs, and drives developments in protein engineering, synthetic biology and rational drug and vaccine design. As demonstrated in this thesis, a detailed understanding of protein-ligand binding can be achieved by combining biophysical, structural, computational and evolution-based methods to elucidate the relationship between the structure and dynamics of the interacting molecules and the thermodynamics, properties (e.g. affinity, kinetics, allostery) and biological consequences of binding events.

In particular, this thesis centers around using X-ray crystallography and other biophysical methods, such as isothermal titration calorimetry, to understand the structural factors that govern several diverse molecular recognition events. In each section, I combine an understanding of molecular evolution with experimental methods to characterise how the function of these proteins is influenced by changes in their sequence, structure and structural flexibility. We discuss the implications that these findings have on protein engineering, medicine, and synthetic biology. In doing so, we not only expand our understanding of how these particular proteins function, but also provide insight into how their activity could be modified, engineered or targeted for useful applications.

Chapter 1 provides an introduction to the concepts and ideas that are central to this thesis and explores how this work fits into our current understanding of the process of molecular recognition. Chapter 2 explores the structural and biophysical properties that govern the function of a PII-like protein that is involved in regulating bicarbonate transport in cyanobacteria. Chapter 3 presents work in which we discuss the evolution of an enzyme from an ancestral solute-binding protein that was specialised for a non-catalytic role, with a focus on the historical changes in structure and dynamics that were required for the emergence and enhancement of the new catalytic activity. In Chapter 4, we characterise an unusual mode of binding between neutralising antibodies and the Plasmodium falciparum circumsporozoite protein and discuss the implications that this has on efforts to design an improved malaria vaccine. In Chapter 5, we explore interactions between molecules involved in nonribosomal peptide synthesis, highlighting the structural basis for specificity and affinity. In Chapter 6, I present a short review that outlines current evolutionary and structural approaches for designing genetically-encoded biosensors for small molecules. This review brings together concepts discussed in the earlier chapters and recapitulates how an understanding of the evolutionary drivers and structural determinants of molecular recognition and protein function can be applied to guide the design of useful molecular tools. Finally, I conclude this thesis with a discussion on recent developments in areas related to the projects and aspects that could to be addressed in future work.