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，酪氨酸侧链在限制性区域内被酶氧化成活性中间体后，自发形成双酪氨酸交联物。这项研究的总体目标是开发一种新的位点特异的蛋白质固定化化学，类似于天然的双赖氨酸交联机制。在温和的氧化剂存在下，人工合成的金属配体和基因上附加在蛋白质上的金属结合肽之间的金属络合物将催化战略放置的酚基(例如酪氨酸)之间的交联键。具体而言，将通过以下方式实现这一目标：i.)多肽金属结合配体组合文库的固相合成，II.通过以下功能快速筛选珠粒文库 标记的含酪氨酸的模型多肽，发现了催化形成双酪氨酸的三元金属络合物，III。)通过在正参数空间内的精细搜索来优化催化活性引线，以及i.)测试与模型蛋白质的活性复合体，以证明自固定蛋白质的效用。与现有的蛋白质修饰方法相比，建议的化学方法可能具有重大优势。首先，建议的方法不依赖于可扩散的试剂，该试剂与特定类别的亲核官能团的所有可访问成员反应。相反，蛋白质修饰将定位于由三元金属络合物预先形成所确定的特定位置。其次，蛋白质修饰位点是由基因决定的，消除了翻译后修饰的需要，并允许从复杂混合物中固定特定的蛋白质。一个重要的健康相关应用程序 建议的技术之一将是更有效地将蛋白质固定到固体载体上的阵列中。通过新的有效的蛋白质修饰技术，蛋白质阵列对临床诊断和其他人类健康保健领域的广泛预期的未来影响可能会更快地实现。

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.