Repeat-Proteins; Stability, Folding Kinetics & Evolution

描述(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，蛋白质折叠是基因和其功能之间的桥梁，也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病也是至关重要的，这些疾病要么是由于基本的基因产物无法折叠到它们的自然状态，要么是因为蛋白质错误折叠成非天然的、易于聚集的复合体，形成有毒的低聚物或纤维。这里提出的研究试图利用简化的结构来理解蛋白质的折叠问题，在简化的结构中，一小群二级结构单元(螺旋、链、转角)在线性阵列中重复。重复蛋白的扩展的模块化结构允许结构单元被移除和插入，提供了折叠能量如何沿着多肽链分布的详细图谱。这种直接的能量图谱可以解决关于蛋白质折叠的长期存在的问题，例如协作性的起源和动力学后果，动力学路径的存在和规范。此外，重复单位的结构相似性使不同地区的贡献能够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质结构，ankyrin(a/a)和LRR(？/非？)重复序列来探索协作性的结构起源，协同性在折叠动力学中的作用，以及当定向作用力促进展开时，整体协同性是如何表现出来的。为了严格量化协作性及其结构起源，我们将利用我们和其他团队最近的一项发现，即相同序列的重复可以建立稳定的阵列。这些“共识阵列”将使用“伊辛”统计模型进行分析，该模型将本征能量与最近邻能量进行量化。共识序列变体将被用来解决哪些类型的相互作用导致了我们在这些蛋白质中看到的非凡的协同性。一旦我们有了解决局部和远程相互作用的变体，我们将能够探索协作性如何影响动力学和过渡态系综，在这个过程中开发一个动力学伊辛模型。对这些蛋白质的动力学分析还将提供有关折叠如何在真正的“平坦”环境中进行的洞察力。这些协作性变体还将用于探索解决方案协作性和端到端强制展开之间的关系。与自然(非共识)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、稳定性和折叠之间的关系的持续洞察。研究将结合标准平衡和停流折叠与协作氢交换质谱仪、原子力显微镜和光镊法。与公共卫生相关：包括癌症和阿尔茨海默病在内的大量人类疾病是由蛋白质不能折叠成活跃的形状，或者折叠成错误的形状，导致细胞和组织中毒引起的。这项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。这些规律将帮助我们理解“折叠疾病”的原因，还将提供新的生物材料，可用于诊断甚至最终治疗人类疾病。

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.