ROBUST PROTEIN FROM MUSSEL BYSSUS

贻贝腹侧线是一种特殊类型的无细胞结缔组织。它们由胶原蛋白组成，在几分钟内制成，在角质层的保护下抵抗降解，并具有一系列理想的机械性能，类似于肌腱胶原、弹性蛋白和牵引丝等重要的生物聚合物。这项研究的长期目标是发现BASSUS的力学性质是如何由其分子结构决定的。具体地说，这项拟议的研究试图发现三种天然存在的融合蛋白在字节线中的分布对这些结构恢复初始长度和屈服后弹性系数的能力有什么影响。蛋白质，preCol-NG，preCol-P和preCol-D都具有嵌段共聚结构，其特征是中心胶原域夹在两个侧翼结构域之间，并被富含组氨酸的氨基和羧基末端覆盖。侧翼结构域类似弹性蛋白(preCol-P)和蜘蛛牵引丝(preCol-D)。这项建议的主要目的(方法)是确定a)前胶体在纤维组装过程中如何定向和排列(使用可逆的双功能交联剂)，b)骨架的成熟对前胶体中N-端和C-端多巴残基以及二硫键形成有什么影响(苯肼衍生多巴，然后进行有限消化和MALDI TOF分析)，c)过渡金属是否通过其富含组氨酸的末端使前胶体交联(通过螯合去除金属，然后进行微观机械分析)，以及d)是否可以在原子力显微镜下拉伸单个前胶体分子。这些结果应该与具有广泛机械性能的支架生物材料的设计特别相关。

Mussel byssal threads are an extraordinary type of acellular connective tissue. Composed of collagen, they are made in minutes, resist degradation when protected by a cuticle, and have a range of desirable mechanical properties that resemble those of important biopolymers such as tendon collagen, elastin, and dragline silk. The long range objective of this research has been to discover how the mechanical properties of byssus are determined by its molecular architecture. Specifically, the proposed research attempts to discover what effect the distribution of three naturally occurring fusion proteins in byssal threads has on the ability of these structures to recover both initial length and modulus after yield.The proteins, preCol-NG, preCol-P and preCol-D, are all characterized by a block copolymer structure that consists of a central collagenous domain sandwiched between two flanking domains, and capped by histidine-rich amino- and carboxy-termini. The flanking domains resemble elastin (preCol-P) and spider dragline silk (preCol-D). The major aims (methods) of this proposal are to determine a) how preCols are oriented and aligned during fiber assembly (using reversible bifunctional crosslinkers), b) what effect maturation of byssus has on N- and C-terminal Dopa residues and on disulfide formation in preCols (phenylhydrazine derivatization for Dopa, followed by limited digestion and MALDI TOF analysis), c) whether transition metals function to cross-link preCols through their histidine-rich termini (metal removal by chelation followed by micromechanical analysis);, and d) whether individual preCol molecules can be stretched on an atomic force microscope. The outcome of these should be of particular relevance to the design of scaffolding biomaterials with a wide range of mechanical properties.