Quantifying Heme-Protein Maturation from Ratiometric Fluorescence Lifetime Measurements on the Single Fluorophore in Its GFP Fusion.

JOURNAL OF PHYSICAL CHEMISTRY A(2020)

引用 3|浏览3
暂无评分
摘要
Protein maturation by heme insertion is a common post-translation modification of key biological importance. Nonetheless, where and when this maturation occurs in eukaryotic cells remain unknown for most heme proteins. Here, we demonstrate for the first time that the maturation of a chromosomally expressed, endogenous heme protein fused to a green fluorescent protein (GFP) can be tracked in live cells. Selecting yeast cytochrome c peroxidase (Ccp1) as our model heme-binding protein, we first characterized the emission in vitro of recombinant Ccpl-GFP with GFP fused C -terminally to Ccp1 by the linker GRRIPGLIN. Time -correlated single -photon counting reveals a single fluorescence lifetime for heme-free apoCcpl-GFP, tau(0) = 2.84 +/- 0.01 ns. Herne bound to Ccp1 only partially quenches GFP fluorescence since holoCcpl GFP exhibits two lifetimes, tau(1) = 0.95 +/- 0.02 and tau(2) = 2.46 +/- 0.03 ns with fractional amplitudes a(1) = 38 +/- 1.5% and a(2) = 62 +/- 1.5%. Also, a and a are independent of Ccp1-GFP concentration and solution pH between 5.5 and 8.0, and a standard plot of al vs % holoCcpl GFP in mixtures with apoCcp1-GFP is linear, establishing that the fraction of Ccpl GFP with heme bound can be determined from a(1). Fluorescence lifetime imaging microscopy (FLIM) of live yeast cells chromosomally expressing the same Ccp1-GFP fusion revealed 30% holoCcpl GFP (i.e., mature Ccp1) and 70% apoCcpl-GFP in agreement with biochemical measurements on cell lysates. Thus, ratiometric fluorescence lifetime measurements offer promise for probing heme-protein maturation in live cells, and we can dispense with the reference fluorophore required for ratiometric intensity-based measurements.
更多
查看译文
AI 理解论文
溯源树
样例
生成溯源树,研究论文发展脉络
Chat Paper
正在生成论文摘要