Nanomechanics of Spiral Proteins

螺旋蛋白的纳米力学

• 批准号: 8042698
• 负责人: PIOTR E MARSZALEK
• 金额: $ 26.76万
• 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 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8042698
• 关键词: Address; Amino Acids; Anemia; Anions; Ankyrin Repeat; Ankyrins; Atomic Force Microscopy; Biological; Elasticity; Engineering; Erythrocyte Membrane; Erythrocytes; Goals; Hemolytic Anemia; Hereditary Spherocytosis; Human; Individual; Inherited; Length; Life; Link; Measurement; Mechanics; Mediating; Medical; Modeling; Molecular; Mutation; Nanotechnology; Play; Positioning Attribute; Principal Investigator; Property; Protein Engineering; Proteins; Research; Role; Shapes; Site-Directed Mutagenesis; Spectrum Analysis; Spherocytes; Structure; Techniques; Tensile Strength; Testing; Work; X-Ray Crystallography; alpha helix; base; cost; graduate student; human disease; in vivo; membrane skeleton; molecular dynamics; mutant; nanomechanical; nanomechanics; nanoscience; programs; 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）：在数千种蛋白质中鉴定出的Ankyrin（ANK）重复序列由成对的反平行字母组成，它们彼此堆叠并形成具有暗示性弹簧的超级螺旋状域具有具有暗示性的弹簧样特性，其主要功能是介导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，并包含形成螺旋结构域的24个ANK重复。 Ankyrin-R可以稳定红细胞膜，ANK重复的突变记录在遗传性球细胞增多症（HS）（HS），即威胁生命的人类贫血。我们最近使用原子力显微镜（AFM）检查了24个Ankyrin-B重复的机械性能，并发现它们的行为非常强，弹性均匀。然而，目前对野生型arnkyrin重复及其突变体的纳米力学特性的分子机制目前尚不了解。该应用的长期目标是阐明管理ANK重复蛋白机械性能的分子机制，并测试以下假设：Ankyrin-R-R的Ank重复序列中与HS相关的突变会损害其非溶剂的特性，这又导致了红细胞转化为spherocytes spherocytess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。由于闭环蛋白的长度仅约10 nm，因此其弹性的测量值具有挑战性，需要使用诸如AFM之类的纳米技术工具，即允许在几乎在体内条件下操纵单分子。在AIM 1中，我们将将蛋白质工程技术与基于AFM的单分子力光谱相结合，以确定Ankyrin-B的纳米弹簧特性是否与Ankyrin-R和其他与结构相关的ANK重复蛋白有关。在AIM 2中，我们将使用位置定向的诱变和AFM直接评估H277R和V463I突变在arnkyrin-R中引起球形细胞增多症的纳米力学性质的影响。在AIM 3中，我们将设计合成ANK重复蛋白和许多ANK重复突变体，将通过X射线晶体学，CD光谱，分子动力学模拟和AFM进行检查，以识别负责螺旋形状的氨基酸，稳定性，张力强度，张力强度和外向/折叠/折叠型蛋白质的蛋白质。这项研究将进一步了解ANK重复蛋白的结构与其纳米力学之间的关系。对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细胞增多症的潜在机械原因，这是一种重要的人类疾病。因此，我们的项目集成了纳米科学和纳米技术方法，以解决重要的生物学和医学问题。在数千种蛋白质中鉴定出Ankyrin（ANK）重复序列，它们在稳定红细胞膜中起着重要作用。 ANK重复中已知的突变记录在遗传性球细胞增多症（HS）中，这是人类中最常见的，威胁生命的遗传性疾病贫血。这项研究利用了原子力显微镜来直接测量ANKYRIN重复的弹性特性，将进一步了解我们对这些蛋白质结构及其春季样特性之间关系的理解。

项目成果

Improving single molecule force spectroscopy through automated real-time data collection and quantification of experimental conditions.

通过自动实时数据收集和实验条件量化改进单分子力谱。

• DOI: 10.1016/j.ultramic.2013.07.020
• 发表时间: 2014
• 期刊: Ultramicroscopy
• 影响因子: 2.2
• 作者: Scholl,ZackaryN;Marszalek,PiotrE
• 通讯作者: Marszalek,PiotrE

Atomic force microscopy captures folded ribosome bound nascent chains.

原子力显微镜捕获折叠的核糖体结合的新生链。

• DOI: 10.1039/c2cc35551e
• 发表时间: 2012
• 期刊: Chemical communications (Cambridge, England)
• 作者: Loksztejn,Anna;Scholl,Zackary;Marszalek,PiotrE
• 通讯作者: Marszalek,PiotrE

Effects of ligand binding on the mechanical properties of ankyrin repeat protein gankyrin.

配体结合对锚蛋白重复蛋白 gankyrin 机械特性的影响。

• DOI: 10.1371/journal.pcbi.1002864
• 发表时间: 2013
• 期刊: PLoS computational biology
• 影响因子: 4.3
• 作者: Settanni,Giovanni;Serquera,David;Marszalek,PiotrE;Paci,Emanuele;Itzhaki,LauraS
• 通讯作者: Itzhaki,LauraS

Nanomechanics of streptavidin hubs for molecular materials.

• DOI: 10.1002/adma.201103316
• 发表时间: 2011-12-15
• 期刊: ADVANCED MATERIALS
• 影响因子: 29.4
• 作者: Kim, Minkyu;Wang, Chien-Chung;Benedetti, Fabrizio;Rabbi, Mahir;Bennett, Vann;Marszalek, Piotr E.
• 通讯作者: Marszalek, Piotr E.