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

X-ray reflectivity (XRR) measurements of femtosecond laser-induced transient gratings (TG) are applied to demonstrate the spatio-temporal coherent control of thermally induced surface deformations on ultrafast timescales. Using grazing incidence x-ray diffraction we unambiguously measure the amplitude of transient surface deformations with sub-Å resolution. Understanding the dynamics of femtosecond TG excitations in terms of superposition of acoustic and thermal gratings makes it possible to develop new ways of coherent control in x-ray diffraction experiments. Being the dominant source of TG signal, the long-living thermal grating with spatial period Λ can be canceled by a second, time-delayed TG excitation shifted by Λ/2. The ultimate speed limits of such an ultrafast x-ray shutter are inferred from the detailed analysis of thermal and acoustic dynamics in TG experiments. [5][6][7] or plasmonic [8,9] degrees of freedom. Strain-induced phenomena may be used to discover new material properties and develop new applications, for example the modification of optical and electronic properties in semiconductor nanostructures [10]. Surface acoustic waves (SAWs) are often employed as a source of lattice strain. They can be generated [11] and controlled [12] optically via the excitation of transient gratings (TGs) [13,14]. Recently, these TG-excitations heave been used to probe heat transport in suspended thin films [15] and magneto-elastic coupling in thin nickel films [16][17][18]. Optical excitation of a solid generates not only coherent sound waves but also incoherent thermal strain. Coherent excitations can be controlled in amplitude and phase by series of light pulses in time domain, which is labeled temporal coherent control [19]. The main fraction of the deposited optical energy is stored in incoherent excitations of the lattice, i.e., heat [20,21] which can consequently not be controlled by a temporal sequence of light pulses. This thermal lattice excitation often generates a background which makes is difficult to precisely observe the coherent acoustic signal in purely optical experiments.In this letter we demonstrate, for the first time, the coherent control of incoherent, thermal transient gratings. We apply spatio-temporal coherent control showing that the spatial part of coherent control adds a new degree of freedom to control the amplitude and the phase of a thermally deformed surface. This is clearly a new approach that introduces the concept of spatial coherent control to the dynamics of incoherent excitations on ultrafast time scales, a phenomenon impossible to achieve with temporal coherent control only. We also demonstrate the control of a transient thermal grating on a timescale faster than the oscillation of the simultaneously excited coherent acoustic modes. Our new quantitative method allows for decomposing the coherent and incoherent dynamics in the sample by measuring the amplitude of the surface excursion with sub-Å precision and ≈ 70 ps temporal resolution. The modification of x-ray diffracti...

Properdin stabilizes convertases formed upon activation of the complement cascade within the immune system. The biological activity of properdin depends on the oligomerization state, but whether properdin oligomers are rigid and how their structure links to function remains unknown. We show by combining electron microscopy and solution scattering, that properdin oligomers adopt extended rigid and well-defined conformations which are well approximated by single models of apparent n-fold rotational symmetry with dimensions of 230–360 Å. Properdin monomers are pretzel-shaped molecules with limited flexibility. In solution, properdin dimers are curved molecules, whereas trimers and tetramers are close to being planar molecules. Structural analysis indicates that simultaneous binding through all binding sites to surface-linked convertases is unlikely for properdin trimer and tetramers. We show that multivalency alone is insufficient for full activity in a cell lysis assay. Hence, the observed rigid extended oligomer structure is an integral component of properdin function.

Phytochromes are bilin-containing photoreceptors that are typically sensitive to the red/far-red region of the visible spectrum. Recently, phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Herein, a combination of time-resolved spectroscopy and structural approaches across picosecond to second timescales have been used to map photochemical mechanisms and structural changes in this atypical group of phytochromes. The photochemistry of an orange/far-red light-sensitive algal phytochrome from Dolihomastix tenuilepis has been investigated by using a combination of visible, IR and X-ray scattering probes. The entire photocycle, correlated with accompanying structural changes in the cofactor/protein, are reported. This study identifies a complex photocycle for this atypical phytochrome. It also highlights a need to combine outcomes from a range of biophysical approaches to unravel complex photochemical and macromolecular processes in multi-domain photoreceptor proteins that are the basis of biological light-mediated signalling.