A PixD—PapB Chimeric Protein Reveals the Function of the BLUF Domain C-Terminal α-Helices for Light Signal Transduction

Abstract

Blue light-using flavin (BLUF) proteins form a subfamily of blue light photoreceptors, are found in many bacteria and algae, and are further classified according to their structures. For one type of BLUF-containing protein, e.g. PixD, the central axes of its two C-terminal α-helices are perpendicular to the β-sheet of its N-terminal BLUF domain. For another type, e.g. PapB, the central axes of its two C-terminal α-helices are parallel to its BLUF domain β-sheet. However, the functional significance of the different orientations with respect to phototransduction is not clear. For the study reported herein, we constructed a chimeric protein, Pix0522, containing the core of the PixD BLUF domain and the C-terminal region of PapB, including the two α-helices, and characterized its biochemical and spectroscopic properties. Fourier transform infrared spectroscopy detected similar light-induced conformational changes in the C-terminal α-helices of Pix0522 and PapB. Pix0522 interacts with and activates the PapB-interacting enzyme, PapA, demonstrating the functionality of Pix0522. These results provide direct evidence that the BLUF C-terminal α-helices function as an intermediary that accepts the flavin-sensed blue light signal and transmits it downstream during phototransduction.