Mechanism of chromophore assisted laser inactivation employing fluorescent proteins.
Mechanism of chromophore assisted laser inactivation employing fluorescent proteins.
复制标题
DOI：
10.1021/ac801663y
复制
发表时间：
2009-03-01
影响因子：
7.4
通讯作者：
Jacobson, Ken
中科院分区：
文献类型：
--
作者：
McLean, Mark A.;Rajfur, Zenon;Chen, Zaozao;Humphrey, David;Yang, Bing;Sligar, Stephen G.;Jacobson, Ken
发色团辅助激光灭活 (CALI) 是一种利用靠近目标蛋白的发色团照射来灭活功能的技术。此前，EGFP 介导的 CALI 已被用于在细胞内以时空限定的方式灭活 EGFP 融合蛋白，但灭活机制尚不清楚。为了帮助阐明荧光蛋白 CALI ([FP]-CALI) 介导的蛋白质失活机制，在体外激光照射后测量了纯化的谷胱甘肽-S-转移酶-FP (GST-EXFP) 融合物的活性。单线态氧和自由基猝灭剂以及氧的去除抑制了 CALI，表明活性氧 (ROS) 的参与。在蛋白质浓度较高时，CALI 后的浊度显着增加，表明邻近融合蛋白发生交联，表明远离活性位点的蛋白质表面残基的损坏导致失活。对照实验排除了样品加热作为这些效应的可能原因。与 GST 融合的不同 FP 突变体的 CALI 效率按 EGFP>EYFP>ECFP 的顺序变化，而结合 FlAsH 的 GST 构建体的 CALI 效率明显高于任何测试的 XFP。 XFP 有效性的层次结构很可能反映了 XFP 结构内捕获并导致荧光团和发色团漂白的 ROS 与逃逸影响邻近蛋白质 CALI 的 ROS 之间的平衡。
Chromophore Assisted Laser Inactivation (CALI) is a technique that uses irradiation of chromophores proximate to a target protein to inactivate function. Previously, EGFP mediated CALI has been used to inactivate EGFP-fusion proteins in a spatio-temporally defined manner within cells but the mechanism of inactivation is unknown. To help elucidate the mechanism of protein inactivation mediated by fluorescent protein CALI ([FP]-CALI), the activities of purified Glutathione-S-transferase-FP (GST-EXFP) fusions were measured after laser irradiation in vitro. Singlet oxygen and free radical quenchers as well as the removal of oxygen inhibited CALI, indicating the involvement of a reactive oxygen species (ROS). At higher concentrations of protein, turbidity after CALI increased significantly indicating cross-linking of proximate fusion proteins suggesting that damage of residues on the surface the protein, distant from the active site, results in inactivation. Control experiments removed sample heating as a possible cause of these effects. Different FP mutants fused to GST vary in their CALI efficiency in the order EGFP>EYFP>ECFP, while a GST construct that binds FlAsH results in significantly higher CALI efficiency than any of the XFPs tested. It is likely that the hierarchy of XFP effectiveness reflects the balance between ROS that are trapped within the XFP structure and cause fluorophore and chromophore bleaching and those that escape to effect CALI of proximate proteins.
登录
查看更多内容
影响因子：
21.3
作者：
Rajfur, Z;Roy, P;Jacobson, K
通讯作者：
Jacobson, K
DOI：
10.1073/pnas.91.7.2659
发表时间：
1994-03-29
影响因子：
11.1
作者：
LIAO, JC;ROIDER, J;JAY, DG
通讯作者：
JAY, DG
影响因子：
3.4
作者：
LINDEN, KG;LIAO, JC;JAY, DG
通讯作者：
JAY, DG
影响因子：
46.9
作者：
Tour, O;Meijer, RM;Tsien, RY
通讯作者：
Tsien, RY
DOI：
10.1073/pnas.85.15.5454
发表时间：
1988-08-01
影响因子：
11.1
作者：
JAY, DG
通讯作者：
JAY, DG