Protein structural dynamics of photoactive yellow protein in solution revealed by pump-probe X-ray solution scattering.
Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump-probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 μs to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The reconstructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.
Center for Time-Resolved Diffraction, Department of Chemistry, Graduate School of Nanoscience & Technology (WCU), KAIST, Daejeon, 305-701, Republic of Korea.
Tae Wu
TW
Kim
Jae Hyuk
JH
Lee
Jungkweon
J
Choi
Kyung Hwan
KH
Kim
Luuk J
LJ
van Wilderen
Laurent
L
Guerin
Youngmin
Y
Kim
Yang Ouk
YO
Jung
Cheolhee
C
Yang
Jeongho
J
Kim
Michael
M
Wulff
Jasper J
JJ
van Thor
Hyotcherl
H
Ihee
D001426:Bacterial Proteins / Q000737:chemistry*; D002621:Chemistry / Q000379:methods*; D018360:Crystallography, X-Ray / Q000379:methods; D007700:Kinetics; D008027:Light; D008968:Molecular Conformation; D020283:Photoreceptors, Microbial / Q000737:chemistry*; D025341:Principal Component Analysis; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D012996:Solutions; D012997:Solvents; D014965:X-Rays
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D012996:Solutions; D012997:Solvents; C050162:photoactive yellow protein, Bacteria