Nanomechanics of Spiral Proteins

螺旋蛋白的纳米力学

• 批准号: 7564100
• 负责人: PIOTR E MARSZALEK
• 金额: $ 26.12万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7564100
• 关键词: Address; Amino Acids; Anemia; Anions; Ankyrin Repeat; Ankyrins; Atomic Force Microscopy; Behavior; Biological; Elasticity; Engineering; Erythrocyte Membrane; Erythrocytes; Fingerprint; Goals; Hemolytic Anemia; Hereditary Spherocytosis; Human; Individual; Inherited; Length; Life; Link; Measurement; Measures; Mechanics; Mediating; Medical; Missense Mutation; Molecular; Molecular Biology; Mutation; Nanotechnology; Play; Property; Protein Engineering; Proteins; Research; Role; Shapes; Site-Directed Mutagenesis; Spectrum Analysis; Spherocytes; Structure; Techniques; Tensile Strength; Testing; X-Ray Crystallography; alpha helix; base; human disease; in vivo; membrane skeleton; molecular dynamics; molecular shape; mutant; nanomechanical; nanomechanics; nanoscience; protein protein interaction; protein structure; single molecule; tool

DESCRIPTION (provided by applicant): Ankyrin (ANK) repeats, identified in thousands of proteins, are composed of pairs of antiparallel alpha- helices that stack on top of each other and form super-helical spiral domains with suggestive spring-like properties, whose primary function is to mediate specific protein-protein interactions. For example, ankyrin-R links the anion exchanger in the erythrocyte membrane to the membrane skeleton and contains 24 ANK repeats that form a spiral domain. Ankyrin-R stabilizes the erythrocyte membrane and mutations in ANK repeats are documented in hereditary spherocytosis (HS), the life-threatening human anemia. We recently examined the mechanical properties of 24 ankyrin-B repeats with atomic force microscopy (AFM) and found that they behave as extremely strong and resilient nanosprings. However, nothing is presently known about molecular mechanisms underlying the nanomechanical properties of wild type ankyrin repeats and their mutants. The long term goal of this application is to elucidate the molecular mechanisms governing the mechanical properties of ANK repeat proteins and to test the hypothesis that the HS-related mutations in ANK repeats of ankyrin-R compromise its nonospring properties, which in turn leads to the conversion of erythrocytes to spherocytes. These objectives can only be achieved by directly testing the mechanical properties of individual ANK repeat proteins and their mutants. Because ankyrins are only ~10 nm in length, the measurements of their elasticity are challenging and require the use of nanotechnology tools such as AFM that allows manipulating single molecules under nearly in vivo conditions. In aim 1, we will combine protein engineering techniques with AFM-based single-molecule force spectroscopy to determine whether the nanospring properties of ankyrin-B, are associated with ankyrin-R and other structurally related ANK repeat proteins. In aim 2, we will use site-directed mutagenesis and AFM to directly evaluate the effects of H277R and V463I mutations in ankyrin-R that cause spherocytosis, on its nanomechanical properties. In aim 3, we will engineer synthetic ANK repeat proteins and a number of ANK repeat mutants, which will be examined by X-ray crystallography, CD spectroscopy, molecular dynamics simulations and AFM in order to identify the amino acids that are responsible for the spiral shape, stability, tensile strength and unfolding/refolding properties of ANK repeat proteins. This research will further our understanding of the relationships between the structure of ANK repeat proteins and their nanomechanics. The study of HS-related ANK repeat mutant proteins may also contribute to an increased understanding of the underlying mechanical cause of spherocytosis, an important human disease. Thus, our project integrates nanoscience and nanotechnology approaches to address important biological and medical problems. Ankyrin (ANK) repeats are identified in thousands of proteins and they play an important role in stabilizing the erythrocyte membrane. Known mutations in ANK repeats are documented in hereditary spherocytosis (HS), the most common, life-threatening inherited hemolytic anemia in humans. This research, which exploits atomic force microscopy for direct measurements of the elastic properties of ankyrin repeats, will further our understanding of the relationship between the structure of these proteins and their spring-like properties.

描述（由申请人提供）：锚蛋白（ANK）重复序列在数千种蛋白质中鉴定，由成对的反平行α-螺旋组成，这些螺旋相互堆叠并形成具有暗示性弹簧样性质的超螺旋螺旋结构域，其主要功能是介导特异性蛋白质-蛋白质相互作用。例如，ANK-R将红细胞膜中的阴离子交换剂连接到膜骨架，并包含形成螺旋结构域的24个ANK重复序列。ANK-R稳定红细胞膜，ANK重复序列的突变在遗传性球形红细胞增多症（HS）中有记载，这是一种危及生命的人类贫血。最近，我们用原子力显微镜（AFM）检查了24个锚定-B重复序列的机械性能，发现它们表现为非常坚固和有弹性的纳米弹簧。然而，目前还不知道野生型锚蛋白重复序列及其突变体的纳米力学性质的分子机制。本申请的长期目标是阐明控制ANK重复蛋白的机械性质的分子机制，并测试以下假设：在anklycer-R的ANK重复中的HS相关突变损害其非弹簧性质，这进而导致红细胞转化为球形红细胞。这些目标只能通过直接测试单个ANK重复蛋白及其突变体的机械性能来实现。由于锚蛋白的长度仅为~10 nm，因此其弹性的测量具有挑战性，并且需要使用纳米技术工具，例如AFM，其允许在接近体内条件下操纵单分子。在目标1中，我们将联合收割机蛋白质工程技术与基于AFM的单分子力光谱学相结合，以确定锚蛋白-B的纳米弹簧特性是否与锚蛋白-R和其他结构相关的ANK重复蛋白相关。在目标2中，我们将使用定点诱变和AFM来直接评估引起球形红细胞增多症的H277 R和V463 I突变对ankle-R的纳米力学性质的影响。在目标3中，我们将设计合成ANK重复蛋白和一些ANK重复突变体，这些突变体将通过X射线晶体学，CD光谱学，分子动力学模拟和AFM进行检查，以确定负责ANK重复蛋白的螺旋形状，稳定性，拉伸强度和展开/重折叠特性的氨基酸。本研究将进一步加深我们对ANK重复序列蛋白的结构与其纳米力学之间关系的理解。HS相关的ANK重复突变蛋白的研究也可能有助于增加对球形红细胞增多症（一种重要的人类疾病）的潜在机械原因的理解。因此，我们的项目整合了纳米科学和纳米技术方法，以解决重要的生物和医学问题。锚蛋白（ANK）重复序列存在于数千种蛋白质中，它们在稳定红细胞膜中起重要作用。已知的ANK重复序列突变在遗传性球形红细胞增多症（HS）中有记载，HS是人类最常见的、危及生命的遗传性溶血性贫血。这项研究利用原子力显微镜直接测量锚蛋白重复序列的弹性特性，将进一步加深我们对这些蛋白质结构与其类似弹簧特性之间关系的理解。