Phytochromes are photoreceptor proteins that transmit a light signal from a photosensory region to an output domain. Photoconversion involves protein conformational changes whose nature is not fully understood. Here, we use time-resolved X-ray scattering and optical spectroscopy to study the kinetics of structural changes in a full-length cyanobacterial phytochrome and in a truncated form with no output domain. X-ray and spectroscopic signals on the µs/ms timescale are largely independent of the presence of the output domain. On longer time-scales, large differences between the full-length and truncated proteins indicate the timeframe during which the structural transition is transmitted from the photosensory region to the output domain and represent a large quaternary motion. The suggested independence of the photosensory-region dynamics on the µs/ms timescale defines a time window in which the photoreaction can be characterized (e.g. for optogenetic design) independently of the nature of the engineered output domain.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

Photolysis of iodoform (CHI) in solution has been extensively studied, but its reaction mechanism remains elusive. In particular, iso-iodoform (iso-CHI-I) is formed as a product of the photolysis reaction, but its detailed structure is not known, and whether it is a major intermediate species has been controversial. Here, by using time-resolved X-ray liquidography, we determined the reaction mechanism of CHI photodissociation in cyclohexane as well as the structure of iso-CHI-I. Both iso-CHI-I and CHI radical were found to be formed within 100 ps with a branching ratio of 40:60. Iodine radicals (I), formed during the course of CHI photolysis, recombine nongeminately with either CHI or I. Based on our structural analysis, the I-I distance and the C-I-I angle of iso-CHI-I were determined to be 2.922 ± 0.004 Å and 133.9 ± 0.8°, respectively.

Size-exclusion chromatography coupled with a laboratory-based small-angle X-ray scattering setup is presented and its performance evaluated for studies of various proteins covering a broad range of molecular weights.

Abstract:We studied the transient electron-transfer process in CsCoFe and RbMnFe Prussian Blue analogues by time-resolved X-ray absorption near-edge structure (XANES) and by time-resolved optical spectroscopy. We performed time-resolved studies on CsCoFe nanocrystals dispersed in solution. The XANES results obtained at room temperature clearly evidence Co III (low spin)Fe II →Co II (high spin)Fe III electron transfer between the metal centers through opposite spectral shifts at the Fe and Co