Investigation of Latent Free Energy in Noncovalent Networks

 描述(申请人提供)：蛋白质中的非共价网络(NCNs)在生物功能中起着关键作用：通过NCNs进行的通信有助于配体结合、催化、变构和信号转导，但NCNs促进这些过程的机制尚未得到普遍认识。通过网络进行通信的一种可能机制是通过储存和释放潜在自由能。值得注意的是，NCN中的每个单独的非共价相互作用不一定是优化的；相反，NCN作为一个整体在蛋白质的限制下进行优化。正因为如此，NCNs储存了潜在的自由能。这为蛋白质在接触外部刺激时做出反应提供了热力学推动力，从而导致下游结果。NCN中力平衡的变化导致潜在自由能的释放，但发生这种情况的分子机制尚不清楚。尽管NCNs在生物功能中发挥着关键作用，但在复杂的生物系统中很难研究它们。然而，用于分子识别的简单模型系统通常不能编码足够的复杂性来充分模拟NCN的性质。在这里，我们建议使用两个中等复杂性的类蛋白质超分子组件来研究潜在自由能在非共价网络门控蛋白激活中的作用，包括对结构突变的反应(目标1)和通过诱导配合结合识别客体(目标2)。这些体系包括多肽链和含有多个β转角的多肽大环宿主、链内和链间氢键以及芳香族相互作用，这些都是相互联系的。在链环中，我们将确定NCN的一个部分的特定变化如何被网络的其余部分补偿(或不补偿)，从而提供对潜在自由能存储和释放机制的洞察(目标1)。将通过实验和计算评估网络每个区域中突变的影响，以探索生成的链烷的结构、动力学和热力学。本课程将研究对刺激作出反应的焓和熵机制。主机-客户系统(AIM 2)将用于研究主机中的NCN与客户的结合亲和力的耦合。蛋白质的NCN在结合时的稳定已被提出以提供显著的结合亲和力(即链霉亲和素-生物素)。我们将通过调整NCN在宿主中的稳定性，并通过实验和计算表征其结构和对客户的反应来验证这一假设。总之，这些研究将阐明NCNs在蛋白质激活中利用潜在自由能的机制，并将为蛋白质功能提供一个新的概念模型，并应用于蛋白质设计和抑制剂开发。

 DESCRIPTION (provided by applicant): 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.