Rocky Mountain Laboratories, NIAID, NIH, Laboratory of Persistent Viral Diseases, 903 S. 4th St., Hamilton, MT 59840 (USA); Currently at the Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, T2N 4Z6 (Canada).
Chembiochem. 2013 Sep 2;14(13):1597-610, 1510. doi: 10.1002/cbic.201300255. Epub 2013 Aug 13.
Protein-protein interactions associated with proteolytic processing and aggregation are integral to normal and pathological aspects of prion protein (PrP) biology. Characterization of these interactions requires the identification of amino acid residues involved. The FlAsH/tetracysteine (FlAsH/TC) tag is a small fluorescent tag amenable to insertion at internal sites in proteins. In this study, we used serial FlAsH/TC insertions (TC-scanning) as a probe to characterize sites of protein-protein interaction between PrP and other molecules. To explore this application in the context of substrate-protease interactions, we analyzed the effect of FlAsH/TC insertions on proteolysis of cellular prion protein (PrPsen) in in vitro reactions and generation of the C1 metabolic fragment of PrPsen in live neuroblastoma cells. The influence of FlAsH/TC insertion was evaluated by TC-scanning across the cleavage sites of each protease. The results showed that FlAsH/TC inhibited protease cleavage only within limited ranges of the cleavage sites, which varied from about one to six residues in width, depending on the protease, providing an estimate of the PrP residues interacting with each protease. TC-scanning was also used to probe a different type of protein-protein interaction: the conformational conversion of FlAsH-PrPsen to the prion disease-associated isoform, PrPres. PrP constructs with FlAsH/TC insertions at residues 90-96 but not 97-101 were converted to FlAsH-PrPres, identifying a boundary separating loosely versus compactly folded regions of PrPres. Our observations demonstrate that TC-scanning with the FlAsH/TC tag can be a versatile method for probing protein-protein interactions and folding processes.
与蛋白水解加工和聚集相关的蛋白-蛋白相互作用是朊病毒蛋白（PrP）生物学中正常和病理状态的重要组成部分。这些相互作用的特征需要确定涉及的氨基酸残基。FlAsH/四半胱氨酸（FlAsH/TC）标记是一种可插入蛋白质内部位点的小荧光标记。在这项研究中，我们使用串联 FlAsH/TC 插入（TC 扫描）作为探针来表征 PrP 与其他分子之间的蛋白-蛋白相互作用位点。为了在底物-蛋白酶相互作用的背景下探索这一应用，我们分析了 FlAsH/TC 插入对体外反应中细胞朊病毒蛋白（PrPsen）的蛋白水解和活神经母细胞瘤细胞中 PrPsen 的 C1 代谢片段生成的影响。通过在每个蛋白酶的切割位点上进行 TC 扫描来评估 FlAsH/TC 插入的影响。结果表明，FlAsH/TC 仅在切割位点的有限范围内抑制蛋白酶切割，其宽度因蛋白酶而异，约为一个到六个残基，提供了与每个蛋白酶相互作用的 PrP 残基的估计值。TC 扫描还用于探测另一种类型的蛋白-蛋白相互作用：FlAsH-PrPsen 到朊病毒病相关异构体 PrPres 的构象转换。在残基 90-96 而不是 97-101 处具有 FlAsH/TC 插入的 PrP 构建体被转换为 FlAsH-PrPres，确定了将松散折叠与紧密折叠的 PrPres 区域分开的边界。我们的观察结果表明，FlAsH/TC 标记的 TC 扫描可以成为探测蛋白-蛋白相互作用和折叠过程的通用方法。
