Imaging farnesyl protein transferase using a topologically activated probe.

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Ras-related farnesyl protein transferase (FPT) is a key enzyme in cell signaling.
• Developing specific and sensitive probes for FPT activity is crucial for biological research and drug discovery.

Purpose of the Study:

• To design and synthesize a novel "smart" activatable peptide-based fluorescent probe for FPT detection.
• To evaluate the probe's specificity, sensitivity, and applicability in various detection media and biological systems.

Main Methods:

• Development of a peptide-based probe engineered for fluorescence activation upon farnesylation by FPT.
• Characterization of probe performance using FPT activity assays, determination of binding constants (Kd), and specificity testing against native peptides.
• Validation of probe performance in pure enzyme assays, cell-based assays, and live cell imaging.

Main Results:

• The smart probe exhibits fluorescence emission upon farnesylation by FPT, enabling detection via fluorescence microscopy, plate reader, and optical imaging.
• The probe demonstrated high specificity for FPT with an IC50 of 1.2 μM, significantly better than the native peptide (IC50 = 0.17 μM).
• The probe possesses a strong binding constant (Kd = 26 nM) and shows a 30-fold fluorescent enhancement in live cell assays compared to control peptides.

Conclusions:

• The developed "smart" activatable peptide probe is a highly specific and sensitive tool for detecting FPT activity.
• The probe's ability to function in diverse media and live cells makes it valuable for biochemical and cell-based studies.
• This probe offers a promising platform for FPT research, diagnostics, and potential therapeutic development.