jp9b11901_si_001.pdf (1.41 MB)
Quantifying Heme–Protein Maturation from Ratiometric Fluorescence Lifetime Measurements on the Single Fluorophore in Its GFP Fusion
journal contribution
posted on 2020-01-14, 23:03 authored by Samaneh Dastpeyman, Robert Godin, Gonzalo Cosa, Ann M. EnglishProtein 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
Ccp1–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 apoCcp1–GFP, τ0 = 2.84 ± 0.01 ns. Heme bound to Ccp1 only partially
quenches GFP fluorescence since holoCcp1–GFP exhibits two lifetimes,
τ1 = 0.95 ± 0.02 and τ2 = 2.46
± 0.03 ns with fractional amplitudes a1 = 38 ± 1.5% and a2 = 62 ±
1.5%. Also, τ and a are independent of Ccp1–GFP
concentration and solution pH between 5.5 and 8.0, and a standard
plot of a1 vs % holoCcp1–GFP in
mixtures with apoCcp1–GFP is linear, establishing that the
fraction of Ccp1–GFP with heme bound can be determined from a1. Fluorescence lifetime imaging microscopy
(FLIM) of live yeast cells chromosomally expressing the same Ccp1–GFP
fusion revealed 30% holoCcp1–GFP (i.e., mature Ccp1) and 70%
apoCcp1–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.
History
Usage metrics
Categories
Keywords
holoCcpGFP Fusion Protein maturationratiometric intensity-based measurementsyeast cytochrome c peroxidaseratiometric fluorescence lifetime measurements offer promisemodel heme-binding proteinquenches GFP fluorescenceFLIMGRRIPGLINapoCcpyeast cells chromosomallyRatiometric Fluorescence Lifetime MeasurementsCcp 1Fluorescence lifetime imaging microscopy
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC