Investigations into Multivalent Ligand Binding Thermodynamics

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2015

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Watts, Brian Edward

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Toone, Eric J

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Abstract

Virtually all biologically relevant functions and processes are mediated by non-covalent, molecular recognition events, demonstrating astonishingly diverse affinities and specificities. Despite extensive research, the origin of affinity and specificity in aqueous solution - specifically the relationship between ligand binding thermodynamics and structure - remains remarkably obscure and is further complicated in the context of multivalent interactions. Multivalency describes the combinatorial interaction of multiple discrete epitopes across multiple binding surfaces where the association is considered as the sum of contributions from each epitope and the consequences of multivalent ligand assembly. Gaining the insight necessary to predictably influence biological processes with novel therapeutics begins with an understanding of the molecular basis of solution-phase interactions, and the thermodynamic parameters that follow from those interactions. Here we continue our efforts to understand the basis of aqueous affinity and the nature of multivalent additivity.

Multivalent additivity is the foundation of fragment-based drug discovery, where small, low affinity ligands are covalently assembled into a single high affinity inhibitor. Such systems are ideally suited for investigating the thermodynamic consequences of multivalent ligand assembly. In the first part of this work, we report the design and synthesis of a fragment-based ligand series for the Grb2-SH2 protein and thermodynamic evaluation of the low affinity ligand fragments compared to the intact, high affinity inhibitor by single and double displacement isothermal titration calorimetry (ITC). Interestingly, our investigations reveal positively cooperative multivalent additivity - a binding free energy of the full ligand greater than the sum of its constituent fragments - that is largely enthalpic in origin. These results contradict the most common theory of multivalent affinity enhancement arising from a "savings" in translational and rotational entropy. The Grb2-SH2 system reported here is the third distinct molecular system in which we have observed enthalpically driven multivalent enhancement of affinity.

Previous research by our group into similar multivalent affinity enhancements in protein-carbohydrate systems - the so-called "cluster glycoside effect" - revealed that evaluation of multivalent interactions in the solution-phase is not straightforward due to the accessibility of two disparate binding motifs: intramolecular, chelate-type binding and intermolecular, aggregative binding. Although a number of powerful techniques for evaluation of solution-phase multivalent interactions have been reported, these bulk techniques are often unable to differentiate between binding modes, obscuring thermodynamic interpretation. In the second part of this work, we report a competitive equilibrium approach to Molecular Recognition Force Microscopy (MRFM) for evaluation of ligand binding at the single-molecule level with potential to preclude aggregative associations. We have optimized surface functionalization strategies and MRFM experimental protocols to evaluate the binding constant of surface- and tip-immobilized single stranded DNA epitopes. Surprisingly, the monovalent affinity of an immobilized species is in remarkable agreement with the solution-phase affinity, suggesting the competitive equilibrium MRFM approach presents a unique opportunity to investigate the nature of multivalent additivity at the single molecule level.

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Watts, Brian Edward (2015). Investigations into Multivalent Ligand Binding Thermodynamics. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/9965.

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