The document describes the design and testing of a fluorescent probe called Amino Phenoxazone Maleimide (APM) to study protein conformational changes. APM was used to label and monitor two membrane proteins: a voltage-gated potassium channel and the beta-2 adrenergic receptor. APM was shown to report on known conformational changes in these proteins and had advantages over other fluorescent probes like having less interference from cellular autofluorescence.

1. A Fluorescent Probe Designed for Studying Protein Conformational Change Cohen, Bruce E. et al . (2005) Proc. Natl. Acad. Sci. USA 102, 965-970

8. Synthesis of APM Fig. 1 Synthesis of APM

9. Fig. 2. Corrected and normalized steady-state fluorescence excitation (A) and emission (B) spectra of the ethanethiol adduct of APM in (from left to right) toluene, ethyl acetate, acetonitrile, ethanol, and water

11. TEV Setup 2 electrodes, V to monitor the membrane potential and I to inject current to hold the membrane at a desired potential. P1 and P2 are salt bridges. High salt solutions, to monitor the solution potential surrounding the oocyte.

12. VCF Profiles Fig. 3. Voltage-clamp fluorometry of Xenopus oocytes expressing Shaker A359C or L361C channels

16. Fig. 4. Fluorescence of purified APM labeled  2 AR in detergent micelles