Cys-Tyr Cofactor in Iron and Copper Proteins

在德克萨斯大学圣安东尼奥分校化学系生命过程化学（CLP）项目的支持下，来自德克萨斯大学圣安东尼奥分校的刘爱民教授正在研究含有半胱氨酸-酪氨酸交联（Cys-Tyr）的蛋白质衍生辅助因子。这些辅助因子是在蛋白质的单向翻译后修饰中产生的。它们具有生物学意义，因为它们在某些酶（如半乳糖氧化酶）中注入催化功能，并增强其他酶（如硫醇双加氧酶）的催化效率。然而，辅助因子合成的化学过程以及Cys-Tyr辅助因子对催化作用的作用尚不清楚。拟开展的研究将集中于阐明辅酶Cys-Tyr交联在酶中形成的机制。参与研究计划的研究生和本科生将学习如何通过晶体学、先进的光谱学和快速发展的用非天然氨基酸取代蛋白质中的氨基酸的技术来确定蛋白质结构。他们还将获得蛋白质催化功能研究方面的专业知识。本研究项目旨在了解铜依赖性半乳糖氧化酶中Cys-Tyr辅助因子的单电子氧化自由基形式的非凡稳定性。这些研究将试图确定酶活性位点的铜中心和芳香残基的自由基自旋密度分布，以及这些分布如何影响催化过程中的质子/电子转移。该研究有望通过表征底物结合复合物和辅因子承载结构，深入了解人硫醇双加氧酶中的Cys-Tyr辅因子如何促进催化。这些研究将利用研究小组已经证明的能力，用非规范氨基酸（ncAAs）基因替代目标氨基酸。虽然策略取代对目标氨基酸残基的改变很小，但它改变了官能团的电子和化学结构。含ncaa的酶变体及其与不同底物的反应将使用化学，光谱和结构生物学方法的组合进行研究。这些研究旨在深入了解不可逆、自加工、C-S键形成以及自由基稳定和铜活性位点氧化还原活性调节的过程。最终，该项目将致力于解决预测蛋白质结构和功能方法的基本挑战，这些方法与当前同类最佳的预测方法不同，并具有协同作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Aimin Liu from the University of Texas at San Antonio is investigating protein-derived cofactors that contain a cysteine-tyrosine crosslink (Cys-Tyr). These cofactors are generated in a unidirectional protein post-translational modification. They have biological significance because they instill catalytic function in some enzymes, such as galactose oxidase, and enhance the catalytic efficiency of other enzymes, such as thiol dioxygenases. However, the chemistry of the cofactor synthesis and how the Cys-Tyr cofactor contributes to catalysis are not well understood. The proposed research will focus on the elucidation of the mechanism by which the Cys-Tyr crosslink of the cofactor is formed in enzymes. The graduate and undergraduate students who are engaged in the research plan will learn how to determine protein structure through crystallography, advanced spectroscopies, and the rapidly developing technology of substitution of an amino acid in a protein with unnatural amino acids. They will also gain expertise in the study of catalytic function in proteins.This research project seeks to understand the extraordinary stability of the one-electron oxidized free radical form of the Cys-Tyr cofactor in copper-dependent galactose oxidase. The proposed studies will seek to determine the radical spin density distributions of the copper center and aromatic residues in the active site of the enzyme and how these distributions affect proton/electron transfer during catalysis. The research is expected to generate insight into how the Cys-Tyr cofactor in human thiol dioxygenases facilitates catalysis by characterization of substrate-bound complexes and cofactor-bearing structures. These studies will leverage the demonstrated ability of the research team to genetically substitute targeted amino acids with noncanonical amino acids (ncAAs). Although the strategic substitution minimally alters the target amino acid residue, it alters the electronic and chemical structure of the functional groups. The ncAA-containing enzyme variants and their reactions with different substrates will be studied using a combination of chemical, spectroscopic, and structural biology methods. These studies aim to provide insight into the irreversible, self-processing, C-S bond formation and the processes by which the radical is stabilized and the redox activity of the copper active site is tuned. Ultimately, the project sets out to contribute to addressing the fundamental challenge of predicting protein structure and function ways that are distinct from and synergistic with current, best-in-class, prediction methods.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.