Functional Implications of Protein Folding Dynamics and Intrinsic Disorder

描述（由申请人提供）：本提案的长期目标是阐明未折叠蛋白质的结构和动态特性以及折叠初始阶段填充的瞬时中间状态。关于这些状态下局部和远程结构倾向的分子位点解析信息对于理解蛋白质的天然结构及其最终功能是如何在氨基酸序列中编码的至关重要。我们将利用溶液核磁共振，包括顺磁弛豫增强和梯度扩散方法，研究溶剂变性葡萄球菌核酸酶和细胞色素c以及内在无序的细胞色素c的构象和动力学性质。这些蛋白质折叠早期的结构形成将通过结合H/D交换、超快速混合和核磁共振分析来监测。通过测量突变对水动力尺寸和弛豫曲线的影响，将确定涉及稳定涉及远程和/或局部相互作用的非随机结构特征的临界残差。这些经验教训和开发的方法将为我们的研究提供一个新的方向，旨在了解模块化多结构域蛋白中内在无序区域的构象性质、动力学和功能意义。内在紊乱在细胞信号传导和癌症相关蛋白中尤其常见，其中单个结构域通常通过长柔性连接体连接。我们将对Na+/H+交换调节因子1 （NHERF1）的结构、动力学和结合特性进行详细的研究，NHERF1是一个信号适配器，由两个属于大PDZ折叠家族的球状结构域、一个c端ezrin结合基序和长无序区组成。广泛的生物物理方法，包括结构和动态核磁共振技术，突变蛋白的热力学分析，使用先进的快速混合技术和计算方法的动力学研究，将被应用。这些发现将为内在无序区域在调节分子内（自抑制）结构域-结构域相互作用和分子间配体相互作用之间的平衡中的作用提供新的见解。最终，我们的工作将有助于更好地理解细胞信号蛋白（如NHERF1）作为分子开关的机制。结合我们对球状蛋白内在无序和化学未展开形式的结构趋势的分析，这些发现将为多肽链中有序和无序之间微妙平衡的序列特征提供新的线索。

DESCRIPTION (provided by applicant): A long-term objective of this proposal is to elucidate the structural and dynamic properties of unfolded proteins and transient intermediate states populated during the initial stages of folding. Molecular site-resolved information on local and long-range structural propensities in these states is critical for understanding how the native structure of a protein, and ultimately its function, are encoded in the amino acid sequence. We will study the conformational and dynamic properties of solvent-denatured staphylococcal nuclease and cytochrome c, as well as the intrinsically disordered apocytochrome c, using solution NMR, including paramagnetic relaxation enhancement and gradient diffusion methods. Structure formation during early stages of folding of these proteins will be monitored by combining H/D exchange with ultra-fast mixing and NMR analysis. Critical residues involved in stabilizing non-random structural features involving long-range and/or local interactions will be identified by measuring the effects of mutations on the hydrodynamic dimensions and relaxation profiles. The lessons learned and approaches developed will benefit a new direction of our research aimed at understanding the conformational properties, dynamics and functional significance of intrinsically disordered regions in modular multidomain proteins. Intrinsic disorder is especially common in cell-signaling and cancer-associated proteins where individual structured domains are often connected via long flexible linkers. We will perform detailed studies of the structure, dynamics and binding properties of Na+/H+ exchanger regulatory factor 1 (NHERF1), a signaling adaptor comprised of two globular domains belonging to the large PDZ fold family, a C-terminal ezrin-binding motif and long disordered regions. A wide array of biophysical approaches, including structural and dynamic NMR techniques, thermodynamic analysis of mutant proteins, kinetic studies using advanced rapid mixing techniques and computational methods, will be applied. The findings will provide new insight into the role of intrinsically disordered regions in regulating the balance between intramolecular (autoinhibitory) domain-domain interactions and intermolecular ligand interactions. Ultimately, our work will lead to a better understanding of the mechanisms by which cell-signaling proteins, such as NHERF1, function as molecular switches. Together with our analysis of the structural tendencies in intrinsically disordered and chemically unfolded forms of globular proteins, the findings will shed new light on the sequence characteristics responsible for the subtle balance between order and disorder in polypeptide chains. PUBLIC HEALTH RELEVANCE: A detailed molecular understanding of the structure and dynamics of unfolded proteins and early stages of folding is critical for establishing how the amino acid sequence determines the native structure of a protein, and ultimately its function. The insight gained will provide a basis for the development of protein-based drugs, and contributes to our mechanistic understanding and treatment of a wide range of diseases that involve aggregation of denatured or misfolded proteins. The proposed structure-function analysis of Na+/H+ exchanger regulatory factor (NHERF1) is highly relevant to human disease, as this protein functions as a signaling adaptor in key cellular pathways implicated in cystic fibrosis, kidney disease, neurological disorders, and cancer.