RUI: Role and Formation of Tyrosine-Cysteine Protein Cofactors

该奖项将调查蛋白质的酪氨酸和半胱氨酸侧链通过芳烷基硫醚键(缩写为Tyr-Cys)进行交联的能力。酪氨酸-半胱氨酸交联物参与氧化催化，由两次质子偶联氧化反应形成。金属-氧化学和正确的侧链构型是形成酪氨酸-半胱氨酸交联剂所必需的。只有四个例子被用X射线结晶学鉴定，这表明了化学、侧链几何形状或检测方法的限制。对蛋白质数据库中所有蛋白质结构的搜索显示，许多蛋白质具有适合形成Tyr-Cys交联键的侧链几何构型。根据酪氨酸形成的条件，在生物条件下，酪氨酸的化学合成应该是容易的。然而，通过X射线结晶学鉴定Tyr-Cys需要仔细分析高分辨率数据。为了更好地鉴定酪氨酸-半胱氨酸的交联物，将采用蛋白质分解和高效液相色谱分离的方法，结合质谱学和核磁共振检测。半胱氨酸双加氧酶含有酪氨酸-半胱氨酸交联键，将用于方法验证。之前没有观察到Tyr-Cys的另外三种蛋白质将被用来测试这些检测方法和之前的蛋白质数据库搜索的组合是否会扩大Tyr-Cys交联链的数量，只是简单地检查静态蛋白质结构。这一信息，以及重新研究Tyr-Cys在胱烯双加氧酶中的功能，将确定Tyr-Cys包含蛋白质的生物学范围是否比目前认为的要广泛得多。这一奖项的广泛影响来自于扩大Tyr-Cys交联物和亲本交联物衍生蛋白辅因子的生物学范围的可能性。此外，该奖项将使用美国国家科学基金会支持的仪器和一个新装修的跨学科实验室。这项工作将为四名本科生化学/生物化学专业的蛋白质化学相关领域的研究生工作做好准备。该奖项还将在一个共享的跨学科研究实验室为多达10名额外的本科生研究人员提供间接培训。两名在科学上代表不足的少数族裔高中生将由国际和平协会和联合国际与ACS项目SEED一起提供指导。

This award will investigate the ability of tyrosine and cysteine sidechains of proteins to crosslink through an arylalkylthioether bond (abbreviated as Tyr-Cys). Tyr-Cys crosslinks participate in oxidation catalysis and are formed by two proton-coupled oxidations. Metal-oxygen chemistry and the correct sidechain geometries are necessary for Tyr-Cys crosslink formation. Only four examples have been identified using X-ray crystallography that suggests limitations from the chemistry, sidechain geometries, or detection methods. A search of all protein structures in the protein databank showed many proteins with appropriate sidechain geometries for Tyr-Cys crosslink formation. Based on conditions for dityrosine formation, the chemistry for Tyr-Cys should be facile under biological conditions. Tyr-Cys identification by X-ray crystallography, however, requires careful analysis of high-resolution data. In order in better identify Tyr-Cys crosslinks a proteolysis and HPLC separation method will be employed using mass spectrometry and nuclear magnetic resonance detection. Cysteine dioxygnease contains a Tyr-Cys crosslink and will be examined for method validation. The three other proteins where Tyr-Cys have not been previously observed will be used to test if the combination of these detection methods and a previous protein databank search will expand the number of Tyr-Cys crosslinks simply examining static protein structure. This information, along with reexamining the function of Tyr-Cys in cystiene dioxygenase, will determine if the biological scope of Tyr-Cys containing proteins is significantly broader than currently thought.Broader impacts of this award come from the potential to expand the biological scope of the Tyr-Cys crosslink and parent crosslinked protein derived cofactors. Additionally, this award will use NSF-supported instrumentation and a newly renovated interdisciplinary laboratory. This work will prepare four undergraduate chemistry/biochemistry majors for graduate work in areas related to protein chemistry. This award will also provide indirect training for up to ten additional undergraduate researchers in a shared interdisciplinary research lab. Two high school students who are underrepresented minorities in the sciences will be mentored by the PI and co-PI in conjunction with ACS Project SEED.