Repeat-Proteins; Stability, Folding Kinetics & Evolution

重复蛋白质；

• 批准号: 7654408
• 负责人: DOUGLAS E. BARRICK
• 金额: $ 27.86万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654408
• 关键词: Address; Adopted; Alzheimer' s Disease; Amino Acid Sequence; Ankyrin Repeat; Ankyrins; Architecture; Atomic Force Microscopy; Biocompatible Materials; Biological Models; Biology; Cations; Cells; Charge; Complex; Consensus; Consensus Sequence; Data; Diagnosis; Disease; Electrostatics; Elements; Environment; Equilibrium; Essential Genes; Evolution; Fiber; Free Energy; Genes; Hand; Height; Hereditary Disease; Hydrogen; Kinetics; Learning; Length; Leucine-Rich Repeat; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Methods; Modeling; Molecular; Nature; Optics; Pathway interactions; Poisoning; Positioning Attribute; Process; Protein Analysis; Protein Array; Proteins; Research; Role; Screening Result; Sequence Analysis; Shapes; Side; Sodium Chloride; Solutions; Statistical Models; Structure; System; Testing; Thermodynamics; Tissues; Variant; density; globular protein; human disease; insight; interfacial; laser tweezer; leucine-rich repeat protein; polypeptide; protein folding; protein misfolding; protein structure; public health relevance; research study; simulation; theories; three dimensional structure

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.

描述（由应用程序提供）：蛋白质折叠是多肽采用其复杂的三维结构的过程。在大多数单体蛋白中，该结构是功能所需的，并且在氨基酸序列中编码。那是蛋白质折叠是基因及其功能之间的桥梁，对于理解生物学是核心。破译蛋白质折叠的规则对于理解无法折叠到其本地状态的基本基因产物或从脱落到非本地，骨骼易用的复合物，形成有毒的寡聚物或纤维的蛋白质而产生的许多通用疾病至关重要。这里提出的研究旨在使用简化体系结构的蛋白质理解折叠问题，在线性阵列中重复了一小部分二级结构单元（Helix，Strand，Turn）的蛋白质。重复蛋白的扩展，模块化结构允许去除和插入结构单位，从而详细绘制了折叠能的分布方式沿多肽链分布。能量景观的直接映射允许解决有关蛋白质折叠的长期问题，例如动力学途径的合作，存在和规范的起源和动力学后果。此外，重复单元的结构相似性允许将不同区域的贡献与明确的贡献进行比较。在这里，我们使用两种不同的重复蛋白结构，分析（A /A）和LRR（€ /non-®）重复探索协调的结构起源，协调在折叠动力学中的作用以及在展开时如何促进散装协调的作用。为了严格量化协调及其结构起源，我们将利用我们和其他群体的最新发现，即可以通过相同序列重复构建稳定阵列。这些“共识阵列”将使用“ ISING”静态模型进行分析，该模型量化了固有的和最近的邻邻能量。共识序列变体将用于解析哪些类型的相互作用会导致我们在这些蛋白质中看到的非凡协调。一旦我们手头上的变体可以解决本地与远程相互作用，我们将能够探讨合作社如何影响动力学和过渡状态集合，从而在此过程中开发动力学模型。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”景观上进行的见解。这些协调变体还将用于探索解决方案协调与端到端强迫展开之间的关系。与天然（非传记）重复阵列的比较将继续深入了解这些简单但无处不在的蛋白质中序列，稳定性和折叠之间的关系。研究将将标准等效的流量折叠与协作氢交换质谱，原子力显微镜和光学镊子方法相结合。公共卫生相关性：包括癌症和阿尔茨海默氏病在内的大量人类疾病是由无法折叠成活性形状的蛋白质引起的，或者折叠成错误的形状，中毒细胞和组织。拟议的研究将使用简化的“重复”蛋白来了解蛋白质如何折叠成独特，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，还将提供可用于诊断甚至最终治疗人类疾病的新生物材料。