Slow Conformational Changes of Blue Light Sensor BLUF Proteins in Milliseconds

Blue light sensor using flavin (BLUF) proteins consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region exhibits a red shift. Ultimately, the light information received in the BLUF domain is transmitted to the functional region. It has been believed that this red shift is complete within nanoseconds. In this study, slow reaction kinetics were discovered in milliseconds (tau(1)- and tau(2)-phase) for all the BLUF proteins examined (AppA, OaPAC, BlrP1, YcgF, PapB, SyPixD, and TePixD). Despite extensive reports on BLUF, this is the first clear observation of the BLUF protein absorption change with the duration in the millisecond time region. From the measurements of some domain-deleted mutants of OaPAC and two chimeric mutants of PixD proteins, it was found that the slower dynamics (tau(2)-phase) are strongly affected by the size and nature of the C-terminal region adjacent to the BLUF domain. Hence, this millisecond reaction is a significant indicator of conformational changes in the C-terminal region, which is essential for the biological functions. On the other hand, the tau(1)-phase commonly exists in all BLUF proteins, including any mutants. The origin of the slow dynamics was studied using site-specific mutants. These results clearly show the importance of Trp in the BLUF domain. Based on this, a reaction scheme for the BLUF reaction is proposed.

Automated summary

The study revealed slow reaction kinetics in milliseconds for all examined BLUF proteins, contrary to previous beliefs of nanosecond red shifts. The slower dynamics in the tau(2)-phase were found to be strongly influenced by the size and nature of the C-terminal region adjacent to the BLUF domain, indicating its importance for biological functions. These results highlight the significance of Trp in the BLUF domain and propose a reaction scheme for BLUF reaction.