CAREER: Characterizing the Mechanism of Copper Exchange in Copper Transfer Proteins

这个职业奖在无机，数学和物理科学理事会化学系的生物无机和有机金属化学项目以及生物科学理事会分子和细胞生物学分部的分子和细胞生物科学项目支持西密歇根大学David Huffman教授的研究，通过确定假定的三坐标的性质来阐明铜的细胞运输的无机化学当金属伴侣（HAH1）将铜转移到Wilson蛋白（WLN4）的靶结构域时形成的中间铜络合物。Ag(I)将用作Cu(I)的光谱探针，因为Ag(I)和Cu(I)的硫化物化学性质相似。这些蛋白的XANES和EXAFS实验表明，当外源硫醇存在时，这些基序可以在二坐标的二边形或三坐标的三角形环境中螯合铜。在核磁共振和x射线结构中，在MXCXXC铜结合基序中发现的蛋氨酸被埋在蛋白质的疏水核心中。使用的方法是：(1)产生每种蛋白质的突变体，其中最不易接近的硫醇发生突变，作为捕获Cu(I)中间体的手段。(2) Cu(I)-HAH1和WLN4之间以及Cu(I)-WLN4和HAH1之间的滴定，将通过UV、XANES/EXAFS和NMR进行监测。类似的实验将用Ag(I)代替Cu(I)，因为Ag(I)配合物不太稳定，109Ag可以作为HAH1/WLN4配合物的核磁共振探针。(3) Cu(I)和Ag(I)模型配合物的合成，作为紫外滴定和XANES实验的标准物。一个挑战将是使铜(I)和银(I)的三角脂肪族硫酸盐配合物。教育部分是在本地和国外扩大对科学领域代表性不足的学生的培训，并使WMU生物化学学生更加了解与科学相关的职业。将采取以下几种方法：(1)与WMU海尼克全球研究所合作，对肯尼亚埃格顿大学的学生进行培训并与教授进行互动。(2)将为卡拉马祖地区的当地高中教师开发讲习班或培训模块，帮助他们向代表性不足的群体推广科学。(3)走进当地的课堂，与当地高中教师合作推广科学。(4)在WMU生物化学导论课程中建立职业论坛。西密歇根大学化学系与当地高中有着长期的合作关系，包括当地的ACS SEED项目、年度高中研讨会和当地的ACS学生和教师奖励项目。主要的重点将是：(1)发展传播职业机会和信息的方法；(2)打破阻碍科学成功的障碍和学生的误解。

This CAREER award in the Inorganic, Bioinorganic and Organometallic Chemistry program in the Division of Chemistry in the Directorate for Mathematics and Physical Sciences and the Molecular and Cellular Biosciences program in the Molecular & Cell BIology Division of the Directorate for Biological Sciences supports research by Professor David Huffman at Western Michigan University to elucidate the inorganic chemistry responsible for the cellular transport of copper by determining the nature of putative three-coordinate intermediate copper complex that forms when the metallochaperone (HAH1) transfers copper to the target domain in the Wilson protein (WLN4). Ag(I) will be used as a spectroscopic probe of Cu(I), since the thiolate chemistry of Ag(I) and Cu(I) are similar. XANES and EXAFS experiments of these proteins show that these motifs can chelate copper in a two-coordinate digonal or a three-coordinate trigonal environment when exogenous thiols are present. In the NMR and X-ray structures, the methionine found in the MXCXXC copper-binding motif is buried in the hydrophobic core of the protein. Methods to be used are: (1) Producing mutants of each protein in which the least accessible thiol is mutated, as a means of trapping the Cu(I) intermediate. (2) Titrations between Cu(I)-HAH1 and WLN4 and also between Cu(I)-WLN4 and HAH1, to be monitored by UV, XANES/EXAFS, and NMR. Similar experiments will replace Cu(I) with Ag(I), since Ag(I) complexes are less labile and 109Ag can be an NMR probe in the HAH1/WLN4 complex. (3) The synthesis of models complexes of Cu(I) and Ag(I) to be used as standards for the UV titrations and XANES experiments. A challenge will be to make trigonal aliphatic thiolate complexes of Cu(I) and Ag(I). The educational component is to expand the training of under-represented students in the sciences, both locally and abroad, and to make WMU biochemistry students more aware of science-related careers. Several approaches will be undertaken: (1) the training of students and interactions with professors from Egerton University in Kenya in collaboration with WMU Haenicke Institute for Global Studies. (2) Workshops or training modules will be developed for local Kalamazoo-area high school teachers to help them promote science to under-represented groups. (3) Going into the local classroom to partner with local high school teachers in the promotion of science. (4) Developing a Career Forum as part of the Introduction to Biochemistry curriculum at WMU. The Department of Chemistry at Western Michigan University (WMU) has a long-standing relationship with area high schools through several venues: the Local ACS SEED Program, the Annual High School Symposium, and the Local ACS Section Awards Program for students and teachers. A primary focus will be (1) to develop methods to disseminate career opportunities and information and (2) to break down the barriers and student misperceptions that hinder success in science.