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，固有无序蛋白质(IDPs)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进细胞信号转导非常重要。IDPs被发现在与膜、小分子或另一种蛋白质结合时折叠。虽然个别国内流离失所者可以以不同的方式折叠，即首先结合一个配体，然后折叠，反之亦然，但确定特定国内流离失所者的这些细节对于试图稳定其复杂的药物并使药物控制其作用将是至关重要的。这项建议旨在研究pKID/KIX复合体，这是从转录因子CREB及其激活因子CREB结合蛋白(CBP)中分离出来的两个结构域。这两个结构域是CREB(通过pKID，IDP)和CBP(通过KIX，配体)联系的地方，只有当CBP与其结合时，CREB才能积极促进下游信号转导。长期以来的研究表明，CREB依赖的信号转导对记忆发育和脑老化很重要，甚至提示这种信号转导可能会受到阿尔茨海默病的影响。因此，为了加深对CREB和CBP之间的关联的了解，希望最终稳定CREB/CBP的关联，并开发药物来促进CREB依赖的信号转导在老年人群中的持续存在，本建议旨在研究pKID与KIX的折叠和结合，以确定复合体形成的分子细节。将使用FRET和基于红外的动力学实验来了解pKID折叠和与KIX结合的详细结构和动力学顺序。更多的实验将表明pKID中的单个残基对折叠有多重要，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对国内流离失所者作为一个整体是如何工作的洞察，以及确定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结合，并可能防止衰老。

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.