Determining protease activity in vivo by fluorescence cross-correlation analysis.

Area of Science:

• Biochemistry
• Cellular Biology
• Biophysics

Background:

• Traditional in vitro biochemical assays lack cellular relevance for studying protein function.
• In vivo analysis is crucial for understanding dynamic processes like protein association/dissociation within living cells.
• Existing in vivo techniques are limited for real-time monitoring of these interactions.

Purpose of the Study:

• To develop and validate a minimally invasive in vivo method for analyzing protein interactions and functions.
• To establish an endogenous fluorescence-based assay avoiding external labeling and delivery.
• To present a model system for studying protease activity in situ.

Main Methods:

• Utilized dual-color fluorescence cross-correlation spectroscopy (FCCS) for in vivo analysis.
• Created double-labeled protease substrates by linking autofluorescent proteins with protease-sensitive linkers.
• Employed genetically encoded autofluorescent protein fusions for endogenous assays.

Main Results:

• Successfully monitored protease reactions in situ using FCCS.
• Demonstrated improved experimental resolution and internal calibration for quantifying protein stability.
• Showcased the ability to assess multiple dynamic parameters simultaneously.

Conclusions:

• The developed in vivo protease assay using FCCS is a promising technique for studying intracellular protein functions.
• This method offers a powerful tool for elucidating dynamic protein-protein interactions in real-time within living cells.
• The approach is easily extendable to study reversible protein-protein interactions.