Self-immobilizing Proteins

在自然界中，蛋白质和其他生物聚合物的高度交联网络是常见的材料。在许多情况下，在酪氨酸侧链被酶促氧化成限制区域内的反应性中间体后，自发地形成双酪氨酸交联。拟议的研究的总体目标是开发一种新的位点特异性蛋白质固定化学类似于天然双酪氨酸交联机制。战略性放置的酚基之间的交联（例如，酪氨酸）将在温和氧化剂存在下被合成金属配体和遗传上附加到蛋白质上的金属结合肽之间的金属络合物催化。具体而言，将通过以下方式实现这一目标：肽金属结合配体的组合文库的固态合成，ii.）快速珠上文库筛选 标记的含酪氨酸的模型肽以发现催化二酪氨酸形成的三元金属络合物，iii.）通过在正参数空间内的精细搜索优化催化活性先导物，和iv.）用模型蛋白测试活性复合物以证明自固定蛋白的效用。所提出的化学方法可能比现有的蛋白质修饰方法具有重大优势。首先，所提出的方法不依赖于与特定类别的亲核官能团的所有可接近成员反应的可扩散试剂。相反，蛋白质修饰将定位于由三元金属络合物的预形成确定的特定位点。其次，蛋白质修饰位点是遗传决定的，消除了翻译后修饰的需要，并允许从复杂的混合物中固定特定的蛋白质。一个重要的健康相关应用 所提出的技术的一个优点是将蛋白质更有效地固定到固体支持物上的阵列中。蛋白质阵列对临床诊断和人类卫生保健的其他领域的广泛预期的未来影响可能会更快地实现新的和有效的蛋白质修饰技术。

In nature, highly crosslinked networks of proteins and other biopolymers are common materials. In many cases, dityrosine crosslinks form spontaneously after tyrosine sidechains are enzymatically oxidized into reactive intermediates within a restricted region. The overall objective of the proposed research is to develop a novel site-specific protein immobilization chemistry similar to natural dityrosine crosslinking mechanisms. Crosslinks between strategicallly placed phenolic groups (e.g., tyrosine) will be catalyzed in the presence of a mild oxidant by a metal complex between a synthetic metal ligand and a metal binding peptide genetically appended to the protein. Specifically, this objective will be pursued by: i.) solid-state synthesis of combinatorial libraries of peptidic metal binding ligands, ii.) rapid on-bead library screening with labelled tyrosine-containing model peptides to discover a ternary metal complex that catalyzes dityrosine formation, iii.) optimization of catalytically active leads through refined searching within the positive parameter space, and iv.) testing of active complexes with model proteins to demonstrate the utility of self-immobilizing proteins. The proposed chemistry may have major advantages over existing protein modification methodologies. First, the proposed method does not rely on diffusible reagents that react with all accessible members of a particular class of nucleophilic functional group. Rather, protein modification will be localized to specific sites determined by the pre-formation of a ternary metal complex. Second, the protein modification site is determined genetically, eliminating the need for post-translational modification and allowing specific protein immobilization from complex mixtures. An important health related application of the proposed technology will be more efficient immobilization of proteins into arrays on solid supports. The widely expected future impact of protein arrays on clinical diagnosis and other areas of human health care may be realized more quickly with new and effective protein modification technology.