scholarly journals Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

2019 ◽  
Author(s):  
Tobias Weinert ◽  
Petr Skopintsev ◽  
Daniel James ◽  
Florian Dworkowski ◽  
Ezequiel Panepucci ◽  
...  
Keyword(s):  
Structural Changes ◽  
Conformational Dynamics ◽  
Proton Pumping ◽  
Uptake Mechanism ◽  
Time Resolved ◽  
X Ray ◽  
Membrane Pump ◽  
Protein Motions ◽  
Hydrophobic Barrier ◽  
Laser Experiments

AbstractConformational dynamics are essential for proteins to function. Here we describe how we adapted time-resolved serial crystallography developed at X-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes upon proton pumping in bacteriorhodopsin over 200 ms in time. The snapshot from the first 5 ms after photoactivation shows structural changes associated with proton release at comparable quality to previous X-ray laser experiments. From 10-15 ms onwards we observe large additional structural rearrangements up to 9 Å on the cytoplasmic side. Rotation of Leu93 and Phe219 opens a hydrophobic barrier leading to the formation of a water chain connecting the intracellular Asp96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

scholarly journals Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. 61-65 ◽  
Author(s):  
Tobias Weinert ◽  
Petr Skopintsev ◽  
Daniel James ◽  
Florian Dworkowski ◽  
Ezequiel Panepucci ◽  
...  
Keyword(s):  
Structural Changes ◽  
Conformational Dynamics ◽  
Uptake Mechanism ◽  
Time Resolved ◽  
X Ray ◽  
Membrane Pump ◽  
Protein Motions ◽  
Hydrophobic Barrier ◽  
Proton Uptake ◽  
Laser Experiments

Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid–96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

scholarly journals Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

2019 ◽  
Vol 116 (51) ◽  
pp. 25634-25640 ◽  
Author(s):  
Medhanjali Dasgupta ◽  
Dominik Budday ◽  
Saulo H. P. de Oliveira ◽  
Peter Madzelan ◽  
Darya Marchany-Rivera ◽  
...  
Keyword(s):  
Active Site ◽  
Enzyme Catalysis ◽  
Conformational Dynamics ◽  
Catalytic Efficiency ◽  
Enzyme Mechanism ◽  
Time Resolved ◽  
X Ray ◽  
Protein Motions ◽  
X Ray Crystallography ◽  
Active Site Cysteine

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

scholarly journals First steps towards time-resolved 'in-house' X-ray diffraction experiments

2014 ◽  
Vol 70 (a1) ◽  
pp. C775-C775 ◽  
Author(s):  
Radoslaw Kaminski ◽  
Jason Benedict ◽  
Elzbieta Trzop ◽  
Katarzyna Jarzembska ◽  
Bertrand Fournier ◽  
...  
Keyword(s):  
Excited State ◽  
Time Resolution ◽  
Structural Changes ◽  
Laser Pulses ◽  
High Repetition Rate ◽  
X Ray Diffraction ◽  
Laboratory Equipment ◽  
Time Resolved ◽  
X Ray ◽  
Ligand Charge Transfer

High-intensity X-ray sources, such as synchrotrons or X-ray free electron lasers, providing up to 100 ps time-resolution allow for studying very short-lived excited electronic states in molecular crystals. Some recent examples constitute investigations of Rh...Rh bond shortening,[1] or metal-to-ligand charge transfer processes in CuI complexes.[2] Nevertheless, in cases in which the lifetime of excited state species exceeds 10 μs it is now possible, due to the dramatic increase in the brightness of X-ray sources and the sensitivity of detectors, to use laboratory equipment to explore structural changes upon excitation. Consequently, in this contribution we present detailed technical description of the 'in-house' X-ray diffraction setup allowing for the laser-pump X-ray-probe experiments within the time-resolution at the order of 10 μs or larger. The experimental setup consists of a modified Bruker Mo-rotating-anode diffractometer, coupled with the high-frequency Nd:YAG laser (λ = 355 nm). The required synchronization of the laser pulses and the X-ray beam is realized via the optical chopper mounted across the beam-path. Chopper and laser capabilities enable high-repetition-rate experiments reaching up to 100 kHz. In addition, the laser shutter is being directly controlled though the original diffractometer software, allowing for collection of the data in a similar manner as done at the synchrotron (alternating light-ON & light-OFF frames). The laser beam itself is split into two allowing for improved uniform light delivery onto the crystal specimen. The designed setup was tested on the chosen set of crystals exhibiting rather long-lived excited state, such as, the Cu2Br2L2 (L = C5H4N-NMe2) complex, for which the determined lifetime is about 100 μs at 90 K. The results shall be presented. Research is funded by the National Science Foundation (CHE1213223). KNJ is supported by the Polish Ministry of Science and Higher Education through the "Mobility Plus" program.

scholarly journals Examining the conformational dynamics of macromolecules with time-resolved synchrotron X-ray ‘footprinting’

Structure ◽  
1997 ◽  
Vol 5 (7) ◽  
pp. 865-869 ◽  
Author(s):  
Mark R Chance ◽  
Bianca Sclavi ◽  
Sarah A Woodson ◽  
Michael Brenowitz
Keyword(s):  
Conformational Dynamics ◽  
Time Resolved ◽  
X Ray

scholarly journals Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

1991 ◽  
Vol 10 (3) ◽  
pp. 521-526 ◽  
Author(s):  
M. H. Koch ◽  
N. A. Dencher ◽  
D. Oesterhelt ◽  
H. J. Plöhn ◽  
G. Rapp ◽  
...  
Keyword(s):  
Diffraction Study ◽  
Structural Changes ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray

scholarly journals A simultaneous multiple angle-wavelength dispersive X-ray reflectometer using a bent-twisted polychromator crystal

2012 ◽  
Vol 20 (1) ◽  
pp. 80-88 ◽  
Author(s):  
Tadashi Matsushita ◽  
Etsuo Arakawa ◽  
Wolfgang Voegeli ◽  
Yohko F. Yano
Keyword(s):  
Structural Changes ◽  
Vertical Direction ◽  
Sample Surface ◽  
Array Detector ◽  
Single Crystal Substrate ◽  
Time Resolved ◽  
Reflectivity Curve ◽  
X Ray ◽  
Monochromator Crystal ◽  
Crystal Substrate

An X-ray reflectometer has been developed, which can simultaneously measure the whole specular X-ray reflectivity curve with no need for rotation of the sample, detector or monochromator crystal during the measurement. A bent-twisted crystal polychromator is used to realise a convergent X-ray beam which has continuously varying energyE(wavelength λ) and glancing angle α to the sample surface as a function of horizontal direction. This convergent beam is reflected in the vertical direction by the sample placed horizontally at the focus and then diverges horizontally and vertically. The normalized intensity distribution of the reflected beam measured downstream of the specimen with a two-dimensional pixel array detector (PILATUS 100K) represents the reflectivity curve. Specular X-ray reflectivity curves were measured from a commercially available silicon (100) wafer, a thin gold film coated on a silicon single-crystal substrate and the surface of liquid ethylene glycol with data collection times of 0.01 to 1000 s using synchrotron radiation from a bending-magnet source of a 6.5 GeV electron storage ring. A typical value of the simultaneously covered range of the momentum transfer was 0.01–0.45 Å−1for the silicon wafer sample. The potential of this reflectometer for time-resolved X-ray studies of irreversible structural changes is discussed.

Following the structural changes during zinc-induced crystallization of charged membranes using time-resolved solution X-ray scattering

Soft Matter ◽  
2011 ◽  
Vol 7 (4) ◽  
pp. 1512-1523 ◽  
Author(s):  
Moshe Nadler ◽  
Ariel Steiner ◽  
Tom Dvir ◽  
Or Szekely ◽  
Pablo Szekely ◽  
...  
Keyword(s):  
Structural Changes ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Charged Membranes ◽  
Induced Crystallization ◽  
Ray Scattering

Dynamical Response of the Electric Double Layer Structure of the DEME-TFSI Ionic Liquid to Potential Changes Observed by Time-Resolved X-ray Reflectivity

2016 ◽  
Vol 230 (4) ◽  
Author(s):  
Wolfgang Voegeli ◽  
Etsuo Arakawa ◽  
Tadashi Matsushita ◽  
Osami Sakata ◽  
Yusuke Wakabayashi
Keyword(s):  
Ionic Liquid ◽  
Double Layer ◽  
Time Scale ◽  
Electric Double Layer ◽  
Structural Changes ◽  
Layer Structure ◽  
Dynamical Response ◽  
Relaxation Processes ◽  
Time Resolved ◽  
X Ray

AbstractThe interface between the N,N-diethyl-N-methyl-N-(2-methoxyethyl)ammonium bis(trifluoromethanesulfonyl)imide (DEME-TFSI) ionic liquid and a gold (111) surface was investigated with time-resolved X-ray reflectivity in order to clarify the dynamics of structural changes of the electric double layer after changing the electrode potential. In the experiment, the potential was switched repeatedly between +1.5 V and −1.5 V every 2 s or every 0.3 s, while measuring the specular X-ray reflectivity. When the potential was switched every 2 s, the time dependence of the reflectivity was different from that of the accumulated charge. This indicates structural relaxation processes that occur on a slower time scale than the acummulation of the charge at the electric double layer.When the potential was switched every 0.3 s, on the other hand, the reflectivity changes followed the evolution of the charge of the electric double layer within the experimental precision, indicating that slow relaxation processes without charge transfer do not contribute significantly to structural changes at this time scale.

scholarly journals Observation of Transient Structural Changes on Hydrogen Absorption Process of LaNi4.75Sn0.25 by Time Resolved X-Ray Diffraction

2015 ◽  
Vol 79 (3) ◽  
pp. 124-130 ◽  
Author(s):  
Akihiko Machida ◽  
Kensuke Higuchi ◽  
Yoshinori Katayama ◽  
Kouji Sakaki ◽  
Hyunjeong Kim ◽  
...  
Keyword(s):  
Hydrogen Absorption ◽  
Structural Changes ◽  
Absorption Process ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray
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