Sequence Directed Crystal Design in the Prismatic Layer of the Mollusk

智力优势：软体动物外壳由两个纳米结构的相对层组成。内部的珍珠状珍珠层由文石晶体形态的碳酸钙组成，并赋予复合材料断裂韧性。外层或棱柱层由单晶碳酸钙的长棱柱组成，方解石改性使其具有抗穿刺性和抗裂纹扩展能力。珍珠层和棱柱层最初都是在贝壳由无定形碳酸钙组成的前体相形成的过程中形成的。这里建议研究棱柱层通过特定的带负电荷的天冬氨酸和富含谷氨酸的蛋白质(统称为Asprich)的中介来组装的详细机制，这些蛋白质被认为指导了矿化过程。这类蛋白质在N端和C端有高度保守的序列。来自N-末端结构域的多肽(片段1)促进径向菱面体晶体排列，而来自C-末端的片段2促进晶体表面的孔洞或空洞。该项目将利用代表片段1和2的合成肽来了解保守序列如何控制晶体生长和设计。特别是，一级序列组织、氨基酸同一性、二级结构和金属离子结合能力与晶体形态选择和生长动力学的相关性将被研究。扫描电子显微镜和原子力显微镜将被用来检查晶体的形态。各种生物物理技术(CD、核磁共振、等温滴定量热法、质谱学、纳米SIMS、X射线衍射、计算机模拟)将提供检查Asprich结构域性能的手段，涉及钙(II)和镁(II)结合热力学、2-和3D结构、表面静电学以及方解石基质中的晶体结合和闭合。与布鲁克海文国家实验室(E.DiMasi)和劳伦斯·利弗莫尔国家实验室(C.Orme)的合作将为该项目提供当地没有的设备。BROADER影响：该项目为研究生提供了一个高度多学科的培训平台，范围从肽合成到各种显微镜，到生物物理表征(从同步辐射X射线衍射到核磁共振、纳米SIMS和等温滴定热量计)。该项目有可能促进对生物矿化的理解，并为仿生复合材料的制造提供模型。PI是纽约科学院高中科学夏令营的参与者，邀请1-2名高中生到实验室做研究实验。他还参与了科学伙伴高中科学教师研究计划，该计划向纽约市高中科学教师介绍暑假期间的研究项目。

INTELLECTUAL MERIT: Mollusk shells consist of two nanostructured, opposing layers. The inner, or pearl-like nacreous, layer consists of calcium carbonate in the aragonite crystal morphology and confers fracture toughness on the composite. The outer, or prismatic, layer consists of long prismatic columns of single crystal calcium carbonate in the calcite modification that confers puncture and crack propagation resistance. Both the nacreous and prismatic layers arise initially during shell development from a precursor phase, consisting of amorphous calcium carbonate. Here it is proposed to investigate the detailed mechanism by which the prismatic layer is assembled through the intermediacy of specific negatively charged aspartic acid and glutamic acid-rich proteins, known collectively as Asprich, thought to direct the mineralization process. Proteins of this class have highly conserved sequences at the N and C termini. A peptide (Fragment 1) from the N-terminal domain promotes radial rhombohedral crystal arrangements, while Fragment 2 from the C-terminus promotes porosities or voids at crystal surfaces. This project will utilize synthetic peptides representing Fragments 1 and 2 to understand how conserved sequences control crystal growth and design. In particular, the correlation of primary sequence organization, amino acid identity, secondary structure, and metal ion binding capacity with crystal morphology selection and growth kinetics will be investigated. Scanning electron and atomic force microscopies will be used to examine crystal morphologies. Various biophysical techniques (CD, NMR, isothermal titration calorimetry, mass spectrometry, nano-SIMS, X-ray diffraction, computer simulation) will provide means to examine the properties of the Asprich domains with regard to Ca(II) and Mg(II) binding thermodynamics, 2- and 3D structure, surface electrostatics, and crystal binding and occlusion within the calcite matrix. Collaborations with Brookhaven National Laboratory (E. DiMasi) and Lawrence Livermore National Laboratory (C. Orme) will provide access to equipment not available locally to the project.BROADER IMPACTS: The project provides a platform for highly multidisciplinary training of graduate students ranging from peptide synthesis, to various microscopies, to biophysical characterization with synchrotron X-ray diffraction to NMR, nano-SIMS, and isothermal titration calorimetry. The project has the potential to advance the understanding of biomineralization and to provide models for biomimetic composite fabrication. The PI is a participant in the New York Academy of Sciences High School Science Summer Camp and invites 1-2 high school students to the lab to do research experiments. He also participates in the Partners in Science high school science teacher research program which introduces NYC high school science teachers to research projects during their summer vacation.