Green fluorescent protein rendered susceptible to proteolysis: Positions for protease-sensitive insertions
Green fluorescent protein rendered susceptible to proteolysis: Positions for protease-sensitive insertions
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DOI：
10.1006/abbi.2001.2537
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发表时间：
2001-10-15
影响因子：
3.9
通讯作者：
Williams, MNV
中科院分区：
文献类型：
--
作者：
Chiang, CF;Okou, DT;Williams, MNV
绿色荧光蛋白 (GFP) 对蛋白水解具有高度抗性，并且在与胰蛋白酶或链霉蛋白酶长时间孵育后仍保持未切割，尽管暴露环中有几个假定的胰蛋白酶和胰凝乳蛋白酶位点。我们通过在位置 157、172 或 189 的环中插入五个氨基酸 IEGRS，使 GFP 对蛋白水解敏感。三个插入突变体的激发和发射最大值与野生型相似，但突变体 Omega 172 和 Omega 189 的量子产率较低，表明荧光团的自由度增加。胰蛋白酶在插入片段定义的独特位点切割每个突变体的天然（折叠）形式。链霉蛋白酶在这些变体中也产生类似的消化模式，但还观察到进一步的蛋白水解，这表明初级切割松弛了 GFP 结构并揭示了以前无法访问的位点。用胰蛋白酶消化后，Omega 189 的荧光几乎没有变化，但用增加的链霉蛋白酶消化后，荧光逐渐降低多达 40%。其他变体的荧光没有受到蛋白酶的显着影响，进一步证实了 GFP 变体的显着稳定性。这些构建体在 GFP 残基 189 周围定义了一个新的构象敏感位点，并表明 GFP 可用于设计蛋白酶敏感分子，以监测体内特定的蛋白水解活性。 (C) 2001 年学术出版社。
The green fluorescent protein (GFP) is highly resistant to proteolysis and remains uncleaved after prolonged incubation with trypsin or pronase despite several putative tryptic and chymotryptic sites in exposed loops. We have rendered GFP sensitive to proteolysis by inserting five amino acids, IEGRS, in loops at position 157, 172, or 189. Excitation and emission maxima of the three insertion mutants were similar to those of wild type, but quantum yields of mutants Omega 172 and Omega 189 were lower, indicating increased freedom of the fluorophore. Trypsin cleaved the native (folded) form of each mutant at a unique site defined by the insert. Pronase also yields similar digestion patterns in these variants, but further proteolysis was also observed, suggesting that the primary cleavage relaxes GFP structure and reveals previously inaccessible sites. Fluorescence of Omega 189 changed little upon digestion with trypsin but decreased progressively by as much as 40% upon digestion with increasing amounts of pronase. Fluorescence of other variants was not affected significantly by the proteases, further confirming the remarkable stabilities of GFP variants. These constructs define a new conformation-sensitive site around residue 189 of GFP and show that GFP may be useful for design of protease-susceptible molecules for monitoring of specific proteolytic activities in vivo. (C) 2001 Academic Press.