Probing the Folding Mechanism of Intrinsically Disordered Proteins: The pKID/KIX

描述（由申请人提供）：在蛋白质折叠领域，内在无序蛋白（IDPs）正在成为一种重要但经常被忽视的分子类别。这些蛋白质在生理条件下是无序的，已被发现占人类蛋白质组的30%，通常是细胞信号分子或DNA结合蛋白。了解这些蛋白质的整体作用机制对于改善细胞信号传导非常重要。发现IDPs在与膜、小分子或另一种蛋白质结合时折叠。虽然单个IDPs可以以不同的方式折叠，即首先结合配体然后折叠，反之亦然，但确定特定IDPs的这些细节对于试图稳定其复杂和时尚的药物以控制其作用至关重要。本课题旨在研究从转录因子CREB及其激活因子CREB结合蛋白（CBP）中分离出的两个结构域pKID/KIX复合物。这两个结构域是CREB（通过pKID， IDP）和CBP（通过KIX，配体）结合的地方，只有当CBP与其结合时，CREB才能积极促进下游信号传导。长期以来的研究表明，creb依赖性信号对记忆发育和大脑衰老很重要，甚至表明这种信号可能会因阿尔茨海默病而受损。因此，为了进一步了解CREB和CBP之间的关联，希望最终稳定CREB/CBP关联，并开发药物来促进老年人持续的CREB依赖性信号传导，本提案旨在研究pKID与KIX的折叠和结合，以确定复合物形成的分子细节。我们将利用FRET和基于红外的动力学实验来了解pKID折叠和结合KIX的详细结构和动力学顺序。进一步的实验将显示pKID中单个残基对折叠的重要性，以及增加pKID的稳定性如何影响其与KIX的结合。这些实验的结果将深入了解IDPs如何作为一个整体起作用，并确定CREB/CBP结合的重要分子特征，然后可以用来人为地改善它们的关联，并可能预防衰老。

DESCRIPTION (provided by applicant): Within the protein folding field, intrinsically disordered proteins (IDPs) are emerging as an important yet often overlooked class of molecules. These proteins, which are disordered under physiological conditions, have been found to make up as much as 30% of the human proteome, and are often cell signaling molecules or DNA binding proteins. Understanding the overall mechanism of action for these proteins will be very important in efforts to improve upon cell signaling. IDPs are found to fold upon association with either a membrane, small molecule, or another protein. Though individual IDPs can fold in different ways, i.e. by binding a ligand first and then folding or vice versa, determining these details for specific IDPs will be of the utmost importance for trying to stabilize their complex and fashion pharmaceutical drugs to control their action. This proposal aims to study the pKID/KIX complex, which are two domains isolated from the transcription factor CREB and its activator CREB binding protein (CBP). These two domains are where CREB (via pKID, the IDP) and CBP (via KIX, the ligand) associate, and only upon CBP binding to it can CREB actively promote downstream signaling. Studies have long shown that CREB-dependent signaling is important for memory development and brain aging, and even suggested that this signaling may be impaired by Alzheimer's. Therefore, to increase understanding of the association between CREB and CBP in the hope of ultimately stabilizing CREB/CBP association and developing drugs to promote continued CREB-dependent signaling in the aging populace, this proposal aims to study the folding and binding of pKID to KIX to determine molecular details of the complex formation. Work will be done using FRET and infrared-based kinetic experiments to understand the detailed structural and kinetic order of pKID folding and binding to KIX. Additional experiments will show how important individual residues within pKID are for folding, and how increasing the stability of pKID affects its binding with KIX. The results of these experiments will provide insight into how IDPs work as a whole, as well as determine important molecular characteristics of CREB/CBP binding, which can then be used to artificially improve their association and possibly forestall aging.