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

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Heme insertion is a crucial post-translational modification for many proteins.
• The location and timing of heme maturation in eukaryotic cells are largely unknown for most heme proteins.

Purpose of the Study:

• To develop and validate a method for tracking heme protein maturation in live eukaryotic cells.
• To quantify the extent of heme incorporation into a model heme protein in real-time within cells.

Main Methods:

• Constructed and characterized a recombinant yeast cytochrome c peroxidase fused to green fluorescent protein (Ccp1-GFP).
• Utilized time-correlated single-photon counting to determine fluorescence lifetimes of apo- and holo-Ccp1-GFP in vitro.
• Applied fluorescence lifetime imaging microscopy (FLIM) to analyze Ccp1-GFP maturation in live yeast cells.

Main Results:

• Fluorescence lifetime measurements distinguished between heme-free (apo) and heme-bound (holo) Ccp1-GFP.
• Established a linear correlation between fractional fluorescence amplitude and the percentage of holo-Ccp1-GFP.
• Quantified 30% mature (holo) and 70% immature (apo) Ccp1-GFP in live yeast cells using FLIM.

Conclusions:

• Ratiometric fluorescence lifetime measurements provide a powerful tool for probing heme-protein maturation in live cells.
• This technique eliminates the need for a reference fluorophore, simplifying measurements.
• The study successfully demonstrated real-time monitoring of endogenous heme protein maturation.