STRUCTURE-BASED ENGINEERING OF AN EFFICIENT INFRARED FLUORESCENT MARKER

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Various organisms can sense light through a large family of signaling proteins known as photoreceptors. Upon absorption of a photon in the appropriate wavelength range, photoreceptors undergo structural changes in the chromophore, an organic pigment embedded in the photosensory module of the protein. Phytochromes are red-light photoreceptors originally discovered in plants and more recently in bacteria. They are unique in their ability to undergo reversible photoconversion between two photoisomerizable states, Pr (red light λmax ~ 700 nm) and Pfr (far-red light λmax ~ 750 nm). The light-activation mechanism involves isomerization around C15=C16 double bond of an open chain tetrapyrrole chromophore, resulting in a flip of its D-ring. Recently, a bacteriophytochrome (Bph) from Deinococcus radiodurands, DrBphP, has been engineered for use as a fluorescent marker in mammalian tissues. In collaboration with Dr. Keith Moffat (The University of Chicago, Chicago IL) and Dr. John Kennis (Vrije Universiteit, Amsterdam, Netherlands), we determined that Bph with unusual photochemistry, RpBphP3 from Rhodopseudomonas palustris, denoted P3 is highly fluorescent. This Bph modulate synthesis of light harvesting complex in combination with a second Bph, RpBphP2 denoted P2. P2 and P3 have the same biliverdin chromophore (BV) and share 52% amino acid sequence identity, yet they have distinct photoconversion properties. P2, similar to classical bacteriophytochromes, alternates between Pr and Pfr states. P3 is unusual since it alternates between Pr and a unique Pnr (near-red light λmax ~ 650 nm) state. We identified factors that determine fluorescence and isomerization quantum yields through the application of ultrafast spectroscopy to wild-type and mutants of P2 and P3. This work provides the basis for structure-based conversion of Bph into an efficient near-IR fluorescent marker.