Effects of FlAsH/tetracysteine (TC) Tag on PrP proteolysis and PrPres formation by TC-scanning.
Effects of FlAsH/tetracysteine (TC) Tag on PrP proteolysis and PrPres formation by TC-scanning.
复制标题
通过 TC 扫描，FLAsH/四半胱氨酸 (TC) 标签对 PrP 蛋白水解和 PrPres 形成的影响。
DOI：
10.1002/cbic.201300255
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Baron,GeraldS
中科院分区：
文献类型：
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作者：
Taguchi,Yuzuru;Hohsfield,LindsayA;Hollister,JasonR;Baron,GeraldS
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.
