Multi-scale Modelling of Allostery in Protein Homodimers

Abstract

Allostery is a form of signalling within biomolecules such that ligand binding to a protein affects its activity at a second site. Allostery was described by early models to be driven by structural changes in the protein. However, more recently there has been increasing evidence that dynamics can contribute to or even drive allostery. The protein studied in this thesis, the Catabolite Activator Protein (CAP), is an allosteric protein homodimer that has been shown to exhibit negatively cooperative binding of the ligand cyclic Adenosine Monophosphate (cAMP) to each of its monomers. Interestingly, CAP is a protein whose allostery is believed to be driven by dynamics rather than a conformational change.

In this thesis, a number of coarse grained models are employed to investigate this dynamic allostery in CAP. One family of models, termed Super Coarse Grained (SCG) models explore the global properties of the dynamics of the CAP dimer that cause it to exhibit negatively cooperative allostery. It is shown through these models that changes in protein interactions can provide a basis for changing cooperativity. A second family of coarse grained models called Elastic Network Models (ENM) are studied. These are used to show that adjusting the interactions between specific residues can affect cooperative binding of cAMP to CAP.

A number of atomistic approaches are also used to study the cAMP-CAP system, including Molecular Dynamics (MD) and Normal Mode Analysis (NMA). The efficacy of using such approaches for studying the thermodynamics of the allostery in CAP is investigated. The motion observed within the protein is also studied closely to identify potential allosteric pathways.

X-ray crystallography and Isothermal Titration Calorimetry (ITC) are finally used to investigate how accurately computational methods can describe the cooperative binding of cAMP to CAP. They are also used to try and determine whether the allostery in CAP can be manipulated experimentally without any observed changes to its structure.