Structural photoactivation of a full-length bacterial phytochrome

Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes

Comparison of glottic views and intubation times in the supine and 25 degree back-up positions

Background: We explored whether positioning patients in a 25° back-up sniffing position improved glottic views and ease of intubation. Methods: In the first part of the study, patients were intubated in the standard supine sniffing position. In the second part, the back of the operating table was raised 25° from the horizontal by flexion of the torso at the hips while maintaining the sniffing position. The best view obtained during laryngoscopy was assessed using the Cormack and Lehane classification and Percentage of Glottic Opening (POGO) score. The number of attempts at both laryngoscopy and tracheal intubation, together with the use of ancillary equipment and manoeuvres were recorded. The ease of intubation was indirectly assessed by recording the time interval between beginning of laryngoscopy and insertion of the tracheal tube. Results: Seven hundred eighty one unselected surgical patients scheduled for non-emergency surgery were included. In the back-up position, ancillary laryngeal manoeuvres, which included cricoid pressure, backwards upwards rightward pressure and external laryngeal manipulation, were required less frequently (19.6 % versus 24. 6 %, p = 0.004). The time from beginning of laryngoscopy to insertion of the tracheal tube was 14 % shorter (median time 24 versus 28 s, p = 0.031) in the back-up position. There was no significant difference in glottic views. Conclusions: The 25° back-up position improved the ease of intubation as judged by the need for fewer ancillary manoeuvres and shorter time for intubation. Trial registration: ClinicalTrials.gov Identifier: NCT02934347 registered retrospectively on 14th Oct 2016

Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump–Probe X-ray Solution Scattering

Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I-1, I-2, and I-3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I-1 intermediate is generated within 100 ps and transforms to the R-like I-2 intermediate with a time constant of 3.2 +/- 0.2 ns. Subsequently, the late, T-like I-3 intermediate is formed via subunit rotation, a decrease in the heme-heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 +/- 120 ns for the fully photolyzed form and 5.6 +/- 0.8 mu s for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I-3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme-heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.1149sciescopu

Magma Chemistry and Tectonic Controls of Volcanic Activity in the Southern Ural Area during Early Carboniferous Time

Early Carboniferous (359.3–323.4 Ma) volcanic complexes are widespread in the Southern Ural tectonic province, a fragment of the western (in present-day coordinates) segment of the Central Asian Orogenic Belt. Here, the Lower Carboniferous sequences crop out within the following N–S-trending tectonic zones (from west to east): Magnitogorsk, Ui River, East Ural, Transural, and Valeryanovka. We describe and discuss the geology and geochemistry of the Early Carboniferous volcanic complexes on the basis of published and newly obtained data, with implications for paleo-tectonic models. The western zones are dominated by bimodal rhyolite-basalt series, with the basalts relatively enriched in Ti and Zr but depleted in Nb. The volcanics of the Valeryanovka zone belong to the typically evolved calc-alkaline series, with the derivatives depleted in Ti and Nb. Almost all of the selected groups of volcanics bear geochemical signatures transitional between those of subduction-related and intraplate igneous rocks. The relative enrichment of the volcanics of the East Ural and Transural Zones may be interpreted as a result of a contribution from asthenospheric mantle and/or from subcontinental lithospheric mantle. The volcanics of the Valeryanovka zone reveal features common to subduction-related series of the Andean type. The data obtained allow us to compare the Early Carboniferous geodynamic settings in the western zones with the modern setting of the Northeastern Pacific, whereas the geodynamic setting of the Valeryanovka zone resembles that at the western margin of South America

Sequential conformational transitions and alpha-helical supercoiling regulate a sensor histidine kinase

Sensor histidine kinases are central to sensing in bacteria and in plants. They usually contain sensor, linker, and kinase modules and the structure of many of these components is known. However, it is unclear how the kinase module is structurally regulated. Here, we use nano- to millisecond time-resolved X-ray scattering to visualize the solution structural changes that occur when the light-sensitive model histidine kinase YF1 is activated by blue light. We find that the coiled coil linker and the attached histidine kinase domains undergo a left handed rotation within microseconds. In a much slower second step, the kinase domains rearrange internally. This structural mechanism presents a template for signal transduction in sensor histidine kinases.Sensor histidine kinases (SHK) consist of sensor, linker and kinase modules and different models for SHK signal transduction have been proposed. Here the authors present nano- to millisecond time-resolved X-ray scattering measurements, which reveal a structural mechanism for kinase domain activation in SHK.Peer reviewe

Photoactivation of <i>Drosophila melanogaster</i> cryptochrome through sequential conformational transitions

Cryptochromes are blue-light photoreceptor proteins, which provide input to circadian clocks. The cryptochrome from Drosophila melanogaster (DmCry) modulates the degradation of Timeless and itself. It is unclear how light absorption by the chromophore and the subsequent redox reactions trigger these events. Here, we use nano- to millisecond time-resolved x-ray solution scattering to reveal the light-activated conformational changes in DmCry and the related (6-4) photolyase. DmCry undergoes a series of structural changes, culminating in the release of the carboxyl-terminal tail (CTT). The photolyase has a simpler structural response. We find that the CTT release in DmCry depends on pH. Mutation of a conserved histidine, important for the biochemical activity of DmCry, does not affect transduction of the structural signal to the CTT. Instead, molecular dynamics simulations suggest that it stabilizes the CTT in the resting-state conformation. Our structural photocycle unravels the first molecular events of signal transduction in an animal cryptochrome

Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions

Cryptochromes are blue-light photoreceptor proteins, which provide input to circadian clocks. The cryptochrome from Drosophila melanogaster (DmCry) modulates the degradation of Timeless and itself. It is unclear how light absorption by the chromophore and the subsequent redox reactions trigger these events. Here, we use nano- to millisecond time-resolved x-ray solution scattering to reveal the light-activated conformational changes in DmCry and the related (6-4) photolyase. DmCry undergoes a series of structural changes, culminating in the release of the carboxyl-terminal tail (CTT). The photolyase has a simpler structural response. We find that the CTT release in DmCry depends on pH. Mutation of a conserved histidine, important for the biochemical activity of DmCry, does not affect transduction of the structural signal to the CTT. Instead, molecular dynamics simulations suggest that it stabilizes the CTT in the resting-state conformation. Our structural photocycle unravels the first molecular events of signal transduction in an animal cryptochrome

Structural photoactivation of a full-length bacterial phytochrome

Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro-and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes

X-ray scattering data for: Sequential conformational transitions and alpha-helical supercoiling regulate a sensor histidine kinase

X-ray solution scattering data collected at BioCARS at APS and cSAXS at SLS respectively. The datasets have been normalized but not scaled to eachother. The solvent heating contribution has not been subtracted and the cSAXS data have not been convoluted with the BioCARS wavelength distribution. The data are provided as comma separated values with the q-vector to the far left and laser pump X-ray probe time-delays as ns (BioCARS) or s (cSAXS) in the first row (not applicable to heat data files)