Bimolecular fluorescence complementation analysis of inducible protein interactions: effects of factors affecting protein folding on fluorescent protein fragment association.
Bimolecular fluorescence complementation analysis of inducible protein interactions: effects of factors affecting protein folding on fluorescent protein fragment association.
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DOI：
10.1016/j.jmb.2009.08.069
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发表时间：
2009-12-04
影响因子：
5.6
通讯作者：
Kerppola TK
中科院分区：
文献类型：
--
作者：
Robida AM;Kerppola TK
双分子荧光互补 (BiFC) 分析可以实现活细胞中蛋白质相互作用的亚细胞位置的可视化。我们使用不同荧光蛋白的片段研究了 BiFC 分析的时间分辨率和定量准确性。我们确定了 BiFC 复合物形成响应 FK506 结合蛋白 (FKBP) 和 FKBP-雷帕霉素结合域 (FRB) 之间雷帕霉素诱导相互作用的动力学。与 FKBP 和 FRB 融合的 YFP 片段在添加雷帕霉素 10 分钟后产生可检测的 BiFC 复合物荧光，并在 8 小时内平均荧光强度增加十倍。与 FKBP 融合的金星荧光蛋白的 N 端片段产生了组成型 BiFC 复合物，其中几个 C 端片段与 FRB 融合。含有金星和 YFP 残基的嵌合 N 端片段产生组成型或诱导型 BiFC 复合物，具体取决于细胞培养的温度。所有测试的荧光蛋白片段半最大诱导 BiFC 复合物形成所需的诱导剂浓度与诱导剂对未修饰的 FKBP 和 FRB 的亲和力一致。 FKBP-FRB相互作用的FK506抑制剂的处理阻止了FKBP和FRB融合形成BiFC复合物，但没有破坏现有的BiFC复合物。添加雷帕霉素之前合成的蛋白质形成 BiFC 复合物，其效率与新合成的蛋白质相同。蛋白质合成抑制剂会减弱 BiFC 复合物的形成，而与它们对融合蛋白合成的影响无关。它们抑制 BiFC 复合物形成的动力学表明它们阻止了荧光蛋白片段的缔合，但不能阻止 BiFC 复合物荧光的缓慢成熟。诱导未折叠蛋白反应的试剂也减少了 BiFC 复合物的形成。这些药物的作用因细胞对蛋白质折叠应激的适应而受到抑制。总之，BiFC 分析能够在活细胞中复合物形成后几分钟内检测蛋白质相互作用，但不允许检测复合物解离。有条件的 BiFC 复合物形成取决于荧光蛋白片段的折叠效率，并且可能受到细胞蛋白折叠环境的影响。
Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. We investigated the temporal resolution and the quantitative accuracy of BiFC analysis using fragments of different fluorescent proteins. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of YFP fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 minutes after rapamycin addition and a ten-fold increase in the mean fluorescence intensity in 8 hours. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and YFP produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment of the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized prior to rapamycin addition formed BiFC complexes with the same efficiency as newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggest that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.
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影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
影响因子：
11.4
作者：
Rackham, O;Brown, CM
通讯作者：
Brown, CM
影响因子：
15
作者：
Magliery, TJ;Wilson, CGM;Regan, L
通讯作者：
Regan, L
DOI：
10.1073/pnas.242594299
发表时间：
2002-11-26
影响因子：
11.1
作者：
Paulmurugan, R;Umezawa, Y;Gambhir, SS
通讯作者：
Gambhir, SS
影响因子：
48
作者：
Remy, Ingrid;Michnick, Stephen W.
通讯作者：
Michnick, Stephen W.