Repeat-Proteins; Stability, Folding Kinetics & Evolution

描述（由申请人提供）：蛋白质折叠是多肽采用其复杂的三维结构的过程。在大多数单体蛋白质中，这种结构是功能所必需的，并在氨基酸序列中编码。因此，蛋白质折叠是基因与其功能之间的桥梁，是理解生物学的核心。破译蛋白质折叠的规则对于理解一些遗传疾病也至关重要，这些疾病要么是由于基本基因产物不能折叠到其天然状态，要么是由于蛋白质错误折叠成非天然的、容易聚集的复合体，形成有毒的低聚物或纤维。本文提出的研究旨在利用简化结构的蛋白质来理解折叠问题，其中二级结构单元（螺旋、链、转）的小簇以线性阵列重复。重复蛋白的扩展模块化结构允许结构单元被移除和插入，提供折叠能量如何沿着多肽链分布的详细映射。这种能量格局的直接映射可以解决关于蛋白质折叠的长期问题，例如协同性的起源和动力学后果，动力学途径的存在和规范。此外，重复单元的结构相似性使得不同区域的贡献可以非常清晰地进行比较。在这里，我们使用两种不同的重复蛋白结构，锚蛋白（a/a）和LRR（¿/非¿）重复来探索协同性的结构起源，协同性在折叠动力学中的作用，以及当定向力促进展开时，体协同性是如何表现出来的。为了严格量化合作及其结构起源，我们将利用我们和其他团队最近的发现，即稳定阵列可以由相同序列的重复构建。这些“共识阵列”将使用“Ising”统计模型进行分析，该模型量化了固有能量与最近邻能量。共识序列变异将用于解决哪些类型的相互作用产生了我们在这些蛋白质中看到的非凡的协同性。一旦我们掌握了解决局部与远程相互作用的变体，我们将能够探索协同性如何影响动力学和过渡态集成，并在此过程中开发动力学Ising模型。这些蛋白质的动力学分析也将提供关于折叠如何在真正的“平坦”景观上进行的见解。这些协同性变体还将用于探索解决方案协同性和端到端强制展开之间的关系。与自然（非一致）重复序列阵列的比较将提供对这些简单但普遍存在的蛋白质的序列，稳定性和折叠之间关系的持续洞察。研究将结合标准平衡和停止流动折叠与协作氢交换质谱，原子力显微镜，光学镊子方法。公共卫生相关性：包括癌症和阿尔茨海默病在内的大量人类疾病是由于蛋白质不能折叠成其活跃形状或折叠成错误形状而导致细胞和组织中毒而引起的。拟议的研究将使用简化的“重复”蛋白质来了解蛋白质如何折叠成独特的、确定的结构的规则。这些规则将帮助我们了解“折叠疾病”的原因，也将提供新的生物材料，可用于诊断，也许最终治疗人类疾病。

DESCRIPTION (provided by applicant): Protein folding is the process by which polypeptides adopt their complex, three dimensional structure. In most monomeric proteins, this structure is required for function, and is encoded in the amino acid sequence. Thus, protein folding is the bridge between the gene and its function, and is central to understanding biology. Deciphering the rules by which proteins fold is also critical for understanding a number of genetic diseases that result either from essential gene products that cannot fold to their native state, or from proteins that misfold to a non-native, aggregation-prone complex, forming toxic oligomers or fibers. The research proposed here seeks to understand the folding problem using proteins of a simplified architecture in which a small cluster of secondary structure units (helix, strand, turn) is repeated in a linear array. The extended, modular architecture of repeat proteins allows units of structure to be removed and inserted, providing a detailed mapping of how folding energy is distributed along the polypeptide chain. This direct mapping of the energy landscape allows long-standing questions about protein folding to be addressed, such as the origin and kinetic consequences of cooperativity, existence and specification of kinetic pathways. In addition, the structural similarity of the repeated units allows the contributions of different regions to be compared with great clarity. Here we use two different repeat protein architectures, the ankyrin (a/a) and LRR (¿/non-¿) repeats to explore the structural origins of cooperativity, the role of cooperativity in folding kinetics, and how bulk cooperativity is manifested when unfolding is promoted by a directed force. To rigorously quantify cooperativity and its structural origins, we will take advantage of a recent discovery by us and by other groups that stable arrays can be built of repeats of identical sequence. These "consensus arrays" will be analyzed using an "Ising" statistical model, which quantifies intrinsic versus nearest-neighbor energies. Consensus sequence variants will be used to resolve which types of interactions give rise to the extraordinary cooperativity we have seen in these proteins. Once we have variants in hand that resolve local versus long-range interactions, we will be able to probe how cooperativity influences kinetics and transition state ensembles, developing a kinetic Ising model in the process. Kinetic analysis of these proteins will also provide insights as to how folding proceeds on a genuinely "flat" landscape. These cooperativity variants will also be used to explore the relationship between solution cooperativity and end-to-end forced unfolding. Comparison to natural (nonconsensus) repeat arrays will provide continued insight into the relationships between sequence, stability, and folding in these simple but ubiquitous proteins. Studies will combine standard equilibrium and stopped flow folding with collaborative hydrogen exchange mass spectrometry, atomic force microscopy, and optical tweezer methods. PUBLIC HEALTH RELEVANCE: A large number of human diseases including cancers and Alzheimer's disease are caused by proteins that cannot fold up to their active shapes, or that fold to the wrong shapes, poisoning cells and tissues. The proposed research will use simplified "repeat" proteins to learn the rules of how proteins fold into unique, well-determined structures. These rules will help us to understand the causes of "folding diseases", and will also provide new biomaterials that can be used to diagnose and perhaps ultimately treat human diseases.