Determining Structural Differences in the Dark and Light States of AppA using Vibrational and Ultrafast Fluorescence Spectroscopy

Abstract

The blue light using flavin (BLUF) domain proteins are a novel class of photosensors that bind flavin noncovalently in order to sense and respond to high intensity blue (450 nm) light. The transcriptional antirepressor AppA, is a BLUF photosensor that utilizes a non‐covalently bound flavin chromophore which is unable to undergo large scale structural change upon light absorption. This is in contrast to most photoreceptors which undergo structural reformations such as trans/cis isomerization upon irradiation. It is of great interest to understand how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is thought to result in alterations in a hydrogen bonding network that surrounds the flavin chromophore on the picosecond time scale. The excited photochemistry of wild‐type AppA and several mutants of AppA in which the hydrogen bonding network has been altered, has been analyzed using ultrafast (100 fs) time resolved fluorescence spectroscopy and picosecond time resolved infrared spectroscopy. Reconstitution of the protein with isotopically‐labeled flavin has permitted unambiguous assignment of the ground and excited state modes associated with the flavin C2=O and C4=O groups, and together the data provide a more detailed understanding of the photoexcitation mechanism.