Nanomechanics of Spiral Proteins

描述(申请人提供)：Ankyrin(ANK)重复序列，在数千种蛋白质中被鉴定，由一对相互堆叠的反平行的α-螺旋组成，形成具有弹簧性质的超螺旋结构域，其主要功能是调节特定的蛋白质-蛋白质相互作用。例如，Ankyrin-R将红细胞膜中的阴离子交换器连接到膜骨架上，并包含24个ANK重复序列，形成螺旋域。Ankyrin-R稳定红细胞膜，ANK重复序列突变在遗传性球形细胞增多症(HS)中被证明，这是一种威胁生命的人类贫血。我们最近用原子力显微镜(AFM)检测了24个Ankyrin-B重复序列的机械性能，发现它们表现为极其强大和弹性的纳米弹簧。然而，目前尚不清楚野生型锚蛋白重复序列及其突变体的纳米机械特性背后的分子机制。这一应用的长期目标是阐明控制ANK重复蛋白机械特性的分子机制，并检验以下假设：Ankyrin-R的ANK重复序列中HS相关的突变损害了其非渗透特性，从而导致红细胞向球形细胞的转化。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Anyrins的长度只有10 nm，因此测量其弹性是具有挑战性的，需要使用纳米技术工具，如AFM，这种工具可以在几乎在体内的条件下操纵单分子。在目标1中，我们将结合蛋白质工程技术和基于AFM的单分子力谱来确定ankyrin-B的纳米弹性特性是否与ankyrin-R和其他结构相关的ANK重复蛋白有关。在目标2中，我们将使用定点突变和原子力显微镜直接评估Ankyrin-R中导致球形红细胞增多的H277R和V463I突变对其纳米机械性能的影响。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，通过X射线结晶学、CD光谱、分子动力学模拟和AFM进行研究，以确定与ANK重复蛋白的螺旋形状、稳定性、拉伸强度和去折叠/重折叠特性有关的氨基酸。这项研究将进一步加深我们对ANK重复蛋白结构与其纳米力学之间关系的理解。对HS相关ANK重复突变蛋白的研究也可能有助于加深对球形细胞增多症的潜在机械原因的理解，球形细胞增多症是一种重要的人类疾病。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。Ankyrin(ANK)重复序列存在于数以千计的蛋白质中，它们在稳定红细胞膜方面发挥着重要作用。遗传性球形细胞增多症(HS)是人类最常见的、威胁生命的遗传性溶血性贫血，ANK重复序列的已知突变在遗传性球状细胞增多症(HS)中有记录。这项研究利用原子力显微镜直接测量Ankyrin重复序列的弹性性质，将进一步了解这些蛋白质的结构和它们的弹簧性质之间的关系。

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.