200 research outputs found
Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase
The radical SAM enzyme, spore photoproduct lyase, requires an H-atom transfer (HAT) pathway to catalyze DNA repair. By rational engineering, we demonstrate that it is possible to rewire its HAT pathway, a first step toward the development of novel catalysts based on the radical SAM enzyme scaffold
Distinct transient structural rearrangement of ionized water revealed by XFEL X-ray pump X-ray probe experiment
Using X-ray free electron laser (XFEL) radiation to conduct an X-ray pump
X-ray probe experiment, we studied strongly ionized water as part of our
ongoing work on radiation damage. After irradiance with a pump pulse with a
nominal fluence of ~ J/cm, we observed for pump-probe delays
of 75 fs and longer an unexpected structural rearrangement, exhibiting a
characteristic length scale of ~9 \r{A}. Simulations suggest that the
experiment probes a superposition of ionized water in two distinct regimes. In
the first, fluences expected at the X-ray focus create nearly completely
ionized water, which as a result becomes effectively transparent to the probe.
In the second regime, out of focus pump radiation produces O and
O ions, which rearrange due to Coulombic repulsion over 10s of fs.
Importantly, structural changes in the low fluence regime have implications for
the design of two-pulse X-ray experiments that aim to study unperturbed liquid
samples. Our simulations account for two key observations in the experimental
data: the decrease in ambient water signal and an increase in low-angle X-ray
scattering. They cannot, however, account for the experimentally observed 9
\r{A} feature. A satisfactory account of this feature presents a new challenge
for theory.Comment: 24 main text pages, 6 supplement pages (30 total), 4 main text
figures, 3 supplemental figures, 2 supplemental table
A genetically encoded photoactivatable Rac controls the motility of living cells
The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties1 or using photoreactive small molecule ligands2. However, this requires use of toxic UV wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (i.e. through microinjection). We have developed a new approach to produce genetically-encoded photo-activatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics3,4. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin5,6, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458 or 473 nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, while PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicron precision7,8. Their mutual regulation remains controversial9, with data indicating that Rac inhibits and/or activates Rho10,11. Rac was shown to inhibit RhoA in living cells, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins
Viscous hydrophilic injection matrices for serial crystallography
Serial (femtosecond) crystallography at synchrotron and X-ray free-electron
laser (XFEL) sources distributes the absorbed radiation dose over all crystals
used for data collection and therefore allows measurement of radiation damage
prone systems, including the use of microcrystals for room-temperature
measurements. Serial crystallography relies on fast and efficient exchange of
crystals upon X-ray exposure, which can be achieved using a variety of
methods, including various injection techniques. The latter vary significantly
in their flow rates – gas dynamic virtual nozzle based injectors provide very
thin fast-flowing jets, whereas high-viscosity extrusion injectors produce
much thicker streams with flow rates two to three orders of magnitude lower.
High-viscosity extrusion results in much lower sample consumption, as its
sample delivery speed is commensurate both with typical XFEL repetition rates
and with data acquisition rates at synchrotron sources. An obvious viscous
injection medium is lipidic cubic phase (LCP) as it is used for in meso
membrane protein crystallization. However, LCP has limited compatibility with
many crystallization conditions. While a few other viscous media have been
described in the literature, there is an ongoing need to identify additional
injection media for crystal embedding. Critical attributes are reliable
injection properties and a broad chemical compatibility to accommodate samples
as heterogeneous and sensitive as protein crystals. Here, the use of two novel
hydroÂgels as viscous injection matrices is described, namely sodium
carbÂoxyÂmethyl cellulose and the thermo-reversible block polymer Pluronic
F-127. Both are compatible with various crystallization conditions and yield
acceptable X-ray background. The stability and velocity of the extruded stream
were also analysed and the dependence of the stream velocity on the flow rate
was measured. In contrast with previously characterized injection media, both
new matrices afford very stable adjustable streams suitable for time-resolved
measurements
crystal and solution structures of the multidomain cochaperone DnaJ
Hsp70 chaperones assist in a large variety of protein-folding processes in the
cell. Crucial for these activities is the regulation of Hsp70 by Hsp40
cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP
hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but
is also known to possess chaperone activity of its own. The first structure of
a complete functional dimeric DnaJ was determined and the mobility of its
individual domains in solution was investigated. Crystal structures of the
complete molecular cochaperone DnaJ from Thermus thermophilus comprising the
J, GF and C-terminal domains and of the J and GF domains alone showed an
ordered GF domain interacting with the J domain. Structure-based EPR spin-
labelling studies as well as cross-linking results showed the existence of
multiple states of DnaJ in solution with different arrangements of the various
domains, which has implications for the function of DnaJ.1\. Auflag
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CAMP@FLASH: an end-station for imaging, electron- and ion-spectroscopy, and pump–probe experiments at the FLASH free-electron laser
The non-monochromatic beamline BL1 at the FLASH free-electron laser facility at DESY was upgraded with new transport and focusing optics, and a new permanent end-station, CAMP, was installed. This multi-purpose instrument is optimized for electron- and ion-spectroscopy, imaging and pump–probe experiments at free-electron lasers. It can be equipped with various electron- and ion-spectrometers, along with large-area single-photon-counting pnCCD X-ray detectors, thus enabling a wide range of experiments from atomic, molecular, and cluster physics to material and energy science, chemistry and biology. Here, an overview of the layout, the beam transport and focusing capabilities, and the experimental possibilities of this new end-station are presented, as well as results from its commissioning
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