Real-time probing of structural transitions of a photoactive protein is challenging owing to the lack of a universal time-resolved technique that can probe the changes in both global conformation and light-absorbing chromophore of the protein. In this work, we combine time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) spectroscopy to investigate how the global conformational changes involved in the photoinduced signal transduction of photoactive yellow protein is temporally and spatially related with the local structural change around the light-absorbing chromophore. In particular, we examine the role of internal proton transfer in developing a signaling state of photoactive yellow protein by employing its E46Q mutant, where the internal proton transfer is inhibited by the replacement of a proton donor. The comparison of TRXSS and TA spectroscopy data directly reveals that the global conformational change of the protein, which is probed by TRXSS, is temporally delayed by tens of microseconds from the local structural change of the chromophore, which is probed by TA spectroscopy. The molecular shape of the signaling state reconstructed from the TRXSS curves directly visualizes the three-dimensional conformations of protein intermeidates and reveals that the smaller structural change in E46Q-PYP than in wt-PYP suggested by previous studies is manifested in terms of much smaller protrusion, confirming that the signaling state of E46Q-PYP is only partially developed compared with that of wt-PYP. This finding provides a direct evidence of how the environmental change in the vicinity of the chromophore alters the conformational change of the entire protein matrix.

Bacteriophytochromes (BphPs) are photoreceptors that regulate a wide range of biological mechanisms via red light–absorbing (Pr)–to–far-red light–absorbing (Pfr) reversible photoconversion. The structural dynamics underlying Pfr-to-Pr photoconversion in a liquid solution phase are not well understood. We used time-resolved x-ray solution scattering (TRXSS) to capture light-induced structural transitions in the bathy BphP photosensory module of Pseudomonas aeruginosa . Kinetic analysis of the TRXSS data identifies three distinct structural species, which are attributed to lumi-F, meta-F, and Pr, connected by time constants of 95 μs and 21 ms. Structural analysis based on molecular dynamics simulations shows that the light activation of PaBphP accompanies quaternary structural rearrangements from an “II”-framed close form of the Pfr state to an “O”-framed open form of the Pr state in terms of the helical backbones. This study provides mechanistic insights into how modular signaling proteins such as BphPs transmit structural signals over long distances and regulate their downstream biological responses.

The structural dynamics of in-cage isomerization of CH2I2 and the unusual structure of the loosely-bound isomer precursor were unveiled with femtosecond X-ray liquidography (solution scattering).