Turkewitz Daniel R, Moghaddasi Saba, Alghalayini Amani, D'Amario Claudia, Ali Hala M, Wallach Michael, Valenzuela Stella M
School of Life Sciences, University of Technology Sydney, Sydney, NSW, 2007, Australia.
ARC Research Hub for Integrated Device for End-user Analysis at Low-levels (IDEAL), Faculty of Science, University of Technology Sydney, NSW, 2007, Australia.
Biochem Biophys Rep. 2021 May 14;26:101015. doi: 10.1016/j.bbrep.2021.101015. eCollection 2021 Jul.
The chloride intracellular ion channel protein (CLIC) family are a unique set of ion channels that can exist as soluble and integral membrane proteins. New evidence has emerged that demonstrates CLICs' possess oxidoreductase enzymatic activity and may function as either membrane-spanning ion channels or as globular enzymes. To further characterize the enzymatic profile of members of the CLIC family and to expand our understanding of their functions, we expressed and purified recombinant CLIC1, CLIC3, and a non-functional CLIC1-Cys24A mutant using a Histidine tag, bacterial protein expression system. We demonstrate that the presence of the six-polyhistidine tag at the amino terminus of the proteins led to a decrease in their oxidoreductase enzymatic activity compared to their non-His-tagged counterparts, when assessed using 2-hydroxyethyl disulfide as a substrate. These results strongly suggest the six-polyhistidine tag alters CLIC's structure at the N-terminus, which also contains the enzyme active site. It also raises the need for caution in use of His-tagged proteins when assessing oxidoreductase protein enzymatic function.
氯离子细胞内离子通道蛋白（CLIC）家族是一组独特的离子通道，它们可以以可溶性蛋白和整合膜蛋白的形式存在。新出现的证据表明，CLIC具有氧化还原酶活性，可能作为跨膜离子通道或球状酶发挥作用。为了进一步表征CLIC家族成员的酶学特征并扩展我们对其功能的理解，我们使用组氨酸标签和细菌蛋白表达系统表达并纯化了重组CLIC1、CLIC3和无功能的CLIC1-Cys24A突变体。我们证明，当使用2-羟乙基二硫化物作为底物进行评估时，与未标记组氨酸的对应物相比，蛋白质氨基末端存在的六聚组氨酸标签导致其氧化还原酶活性降低。这些结果强烈表明，六聚组氨酸标签改变了CLIC在N末端的结构，该末端也包含酶活性位点。这也增加了在评估氧化还原酶蛋白酶功能时使用组氨酸标签蛋白时需要谨慎的必要性。
