ROBUST PROTEIN FROM MUSSEL BYSSUS

贻贝丝是一种特殊的非细胞结缔组织。由胶原蛋白组成，它们在几分钟内制成，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，类似于重要的生物聚合物，如肌腱胶原蛋白，弹性蛋白和牵引丝。这项研究的长期目标是发现足丝的机械性能是如何由其分子结构决定的。具体来说，拟议的研究试图发现三种天然存在的融合蛋白在深海丝中的分布对这些结构在屈服后恢复初始长度和模量的能力有什么影响。这些蛋白质，preCol-NG，preCol-P和preCol-D，都以嵌段共聚物结构为特征，该结构由夹在两个侧翼结构域之间的中央胶原结构域组成，并被富含组氨酸的氨基末端和羧基末端封端。侧翼结构域类似于弹性蛋白（preCol-P）和蜘蛛拖丝（preCol-D）。本提案的主要目的（方法）是确定a）在光纤组装过程中，（使用可逆的双功能交联剂），B）足丝的成熟对N-和C-末端多巴残基以及对preCol中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）过渡金属是否起到通过其富组氨酸末端交联preCol的作用（通过螯合去除金属，然后进行微机械分析）;和d）单个preCol分子是否可以在原子力显微镜上拉伸。这些结果应该是特别相关的支架生物材料的设计与广泛的机械性能。

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.