Folding Mechanism of Intrinsically Disordered Proteins: The pKID/KIX complex

描述（由申请人提供）：在蛋白质折叠领域，内在无序蛋白（IDP）正在成为一类重要但经常被忽视的分子。这些蛋白质在生理条件下是无序的，已经发现占人类蛋白质组的30%，并且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IDP在与膜、小分子或另一种蛋白质缔合时折叠。虽然单个IDP可以以不同的方式折叠，即通过首先结合配体然后折叠或反之亦然，但确定特定IDP的这些细节对于试图稳定其复合物和时尚药物以控制其作用至关重要。本研究的目的是研究pKID/KIX复合物，这是从转录因子CREB及其激活剂CREB结合蛋白（CBP）分离的两个结构域。这两个结构域是CREB（通过pKID，IDP）和CBP（通过KIX，配体）缔合的地方，并且只有当CBP与其结合时，CREB才能积极促进下游信号传导。长期以来，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受到阿尔茨海默氏症的损害。因此，为了增加对CREB和CBP之间关联的理解，希望最终稳定CREB/CBP关联并开发药物以促进老年人群中持续的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。工作将使用FRET和基于红外的动力学实验，以了解详细的结构和动力学顺序的pKID折叠和结合KIX。额外的实验将显示pKID内的单个残基对于折叠的重要性，以及增加pKID的稳定性如何影响其与KIX的结合。这些实验的结果将提供深入了解IDP如何作为一个整体工作，以及确定CREB/CBP结合的重要分子特征，然后可以用来人工改善它们的关联，并可能阻止衰老。

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.