New Fluorescent Readouts for Protein Interactions, Gene Expression, and Membrane Potential

Abstract

Fluorescent chemosensors now exist for most of the major spherical inorganic ions of biological interest, such as H⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, and Cl⁻ (for reviews, see [1–3]). Notable progress [4–6] is finally being made on Zn²⁺, which is important both in many enzymes and in the Zn-fingers of DNA-binding proteins. What major new challenges remain for qualitatively new, biologically relevant fluorescent chemosensors? Obviously organic biochemical analytes are a huge and relatively untapped area. Some years ago we developed a fluorescent protein chemosensor [7,8] for cyclic adenosine 3′,5′-monophosphate (cAMP), the only intracellular second messenger that approaches Ca²⁺ in its ubiquity and importance. This chemosensor consists of cAMP-dependent protein kinase, in which the catalytic subunit is labeled with fluorescein and the regulatory subunit is labeled with rhodamine. Fluorescence resonance energy transfer (FRET) from the fluorescein to the rhodamine occurs in the holoenzyme complex but is disrupted when cAMP binds to the regulatory subunits and dissociates them from the catalytic subunits. This chemosensor has been quite successful in a wide variety of cells, revealing many new aspects about cAMP signaling such as subcellular compartmentation [9–11].