Genetically Encoded Fluorescent Probe for Detection of Heme-Induced Conformational Changes in Cytochrome c

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Cytochrome c (Cytc) is crucial for cellular energy metabolism and apoptosis.
• Cytc activation involves heme binding and interaction with cytochrome c heme lyase (CCHL).
• Existing methods for studying Cytc structure lack physiological relevance and spatiotemporal resolution.

Purpose of the Study:

• To develop a genetically encoded fluorescence method for monitoring Cytc conformational changes in living cells.
• To elucidate the mechanism of heme-induced Cytc folding and its interaction with CCHL.

Main Methods:

• Genetically encoded Förster Resonance Energy Transfer (FRET) using Cerulean and Venus fluorescent proteins fused to Cytc.
• Monitoring FRET amplitude to distinguish between unfolded, intermediate, and native Cytc states.
• Measuring Cytc conformational changes upon heme and CCHL coexpression in living cells.

Main Results:

• Heme binding alone induced a partially folded Cytc state, evidenced by a FRET signal shift.
• High heme concentration and CCHL coexpression promoted the recovery of native Cytc structure.
• Cytc showed weak association with CCHL in the absence of heme.

Conclusions:

• A FRET-based approach successfully elucidated heme-induced Cytc conformational changes with spatiotemporal resolution.
• This method provides insights into Cytc folding mechanisms and interactions in vivo.
• The developed technique is applicable for studying Cytc interactions with small molecules and other proteins in live cells.