Slow conformational changes of blue light sensor BLUF proteins in milliseconds

Abstract

BLUF (blue light sensor using flavin) 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 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. Contrary to this commonly accepted scheme, in this study, slow reaction kinetics were discovered in milliseconds (τ1- and τ2-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 (τ2-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 τ1-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.

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“…Tokonami et al performed transient absorption (TA) measurements on the full-length OaPAC and observed a slow reaction phase in the tens of milliseconds reflecting a conformational change at the C-terminus domain, which is consistent with this result. 34 Time-Dependent Evolution of TRIR Bands. To gain further insights into the structural dynamics of photoactivation, we determined the kinetics for the time-dependent evolution at the wavenumbers of some of the key transients and bleaches in the TRIR spectra (Figure 4).…”

Tackling hysteresis in conformational sampling — how to be forgetful with MEMENTO

“…While tens of microseconds can now be simulated on commercial hardware (rising to hundreds of microseconds with stratification, running multiple boxes in parallel), this is still orders of magnitude shorter than many conformational changes, which often reach into regimes of milliseconds and beyond. [2][3][4] Moreover, to accurately describe conformational dynamics at equilibrium, one needs to observe repeated transitions to obtain good statistics. Enhanced sampling methods can help address this issue in various ways: by adaptive spawning of new trajectories, by adjusting potential energy barriers, or by biasing progress along specific reaction coordinates, termed collective variables (CVs).…”