Noncovalent networks (NCNs) in proteins play a critical role in biological function: communication through NCNs contributes to ligand binding, catalysis, allostery, and signal transduction, but the mechanisms by which NCNs contribute to these processes is not generally recognized. One possible mechanism for communication through a network is via the storage and release of latent free energy. Notably, each individual noncovalent interaction in an NCN is not necessarily optimized; instead, the NCN as a whole is optimized within the constraints of the protein. Because of this, NCNs store latent free energy. This provides the thermodynamic driving force for a protein to respond when it contacts an external stimulus, resulting in a downstream outcome. Changes in the balance of forces in the NCN result in the release of latent free energy, but the molecular mechanisms by which this occurs are poorly understood. Despite the critical role NCNs play in biological function, they are difficult to study in complex biological systems. However, simple model systems for molecular recognition generally do not encode enough complexity to adequately model the properties of a NCN. Herein, we propose to use two protein-like supramolecular assemblies of intermediate complexity to study the role of latent free energy in noncovalent network gated protein actuation, including responses to structural mutations (Aim 1) and recognition of a guest via induced-fit binding (Aim 2). These systems include a peptidic catenane and a peptidic macrocyclic host containing multiple beta-turns, intra- and interstrand H-bonds, and aromatic interactions that are all in communication. In the catenane, we will determine how a specific change in one part of a NCN can be compensated for (or not) by the rest of the network, and thus provide insight into mechanisms for storing and releasing latent free energy (Aim 1). The effect of mutations in each region of the network will be assessed experimentally and computationally to probe the structure, dynamics, and thermodynamics of the resultant catenane. Both enthalpic and entropic mechanisms for responding to a stimulus will be investigated. The host-guest system (Aim 2) will be utilized to investigate the coupling of the NCN in the host to the binding affinity of the guest. Stabilization of a protein's NCN upon binding has been proposed to provide significant enhancement of binding affinities (i.e. streptavidin-biotin). We will test this hypothesis by tunin the stability of the NCN in the host and experimentally and computationally characterizing its structure and response to a guest. Together, these studies will elucidate mechanisms by which NCNs utilize latent free energy in protein actuation and will provide a new conceptual model for protein function with applications to protein design and inhibitor development.

Public Health Relevance

Communication through noncovalent networks (NCNs) contributes a vast range of biological functions important to human health, including ligand binding, catalysis, allostery, and signal transduction, but the molecular mechanisms by which they function are poorly understood. This proposal aims to define such mechanisms using two related models for protein NCNs in folding and binding, which have the advantages of being small enough to study at the molecular level, but complex enough to exhibit protein-like behavior. Molecular level investigations coupled with computational studies aim to provide a new conceptual model for the role of NCNs in protein actuation with applications to functional protein design and inhibitor development.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Synthetic and Biological Chemistry A Study Section (SBCA)
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Fabian, Miles
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University of North Carolina Chapel Hill
Schools of Arts and Sciences
Chapel Hill
United States
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Chung, Mee-Kyung; White, Peter S; Lee, Stephen J et al. (2016) Investigation of a Catenane with a Responsive Noncovalent Network: Mimicking Long-Range Responses in Proteins. J Am Chem Soc 138:13344-13352