Early-stage dynamics of chloride ion–pumping rhodopsin revealed by a femtosecond X-ray laser

Chloride ion–pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl− into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation–diffusion process upon light-triggered retinal isomerization.

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Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions

Science Advances ◽

10.1126/sciadv.aaw1531 ◽

2019 ◽

Vol 5 (7) ◽

pp. eaaw1531 ◽

Cited By ~ 7

Author(s):

Oskar Berntsson ◽

Ryan Rodriguez ◽

Léocadie Henry ◽

Matthijs R. Panman ◽

Ashley J. Hughes ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Conformational Changes ◽

Structural Changes ◽

Structural Response ◽

Biochemical Activity ◽

Time Resolved ◽

X Ray ◽

Molecular Events ◽

Carboxyl Terminal ◽

Dynamics Simulations

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.

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Structure and dynamics of chloride ion pumping rhodopsin revealed by time-resolved SFX and atomic molecular dynamics simulations

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s0108767319096363 ◽

2019 ◽

Vol 75 (a1) ◽

pp. a373-a373

Author(s):

Haiguang Liu ◽

Ji-Hye Yun ◽

Xuanxuan Li ◽

Jae-Hyun Park ◽

Zeyu Jin ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Chloride Ion ◽

Time Resolved ◽

Structure And Dynamics ◽

Dynamics Simulations ◽

Ion Pumping

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Structural changes of tailless bacteriophage ΦX174 during penetration of bacterial cell walls

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716614114 ◽

2017 ◽

Vol 114 (52) ◽

pp. 13708-13713 ◽

Cited By ~ 18

Author(s):

Yingyuan Sun ◽

Aaron P. Roznowski ◽

Joshua M. Tokuda ◽

Thomas Klose ◽

Alexander Mauney ◽

...

Keyword(s):

Conformational Changes ◽

Cell Walls ◽

Structural Changes ◽

The Other ◽

Host Bacterium ◽

Time Resolved ◽

Viral Attachment ◽

X Ray ◽

X Ray Scattering ◽

Cryo Electron Microscopy

Unlike tailed bacteriophages, which use a preformed tail for transporting their genomes into a host bacterium, the ssDNA bacteriophage ΦX174 is tailless. Using cryo-electron microscopy and time-resolved small-angle X-ray scattering, we show that lipopolysaccharides (LPS) form bilayers that interact with ΦX174 at an icosahedral fivefold vertex and induce single-stranded (ss) DNA genome ejection. The structures of ΦX174 complexed with LPS have been determined for the pre- and post-ssDNA ejection states. The ejection is initiated by the loss of the G protein spike that encounters the LPS, followed by conformational changes of two polypeptide loops on the major capsid F proteins. One of these loops mediates viral attachment, and the other participates in making the fivefold channel at the vertex contacting the LPS.

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Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization

IUCrJ ◽

10.1107/s2052252518012149 ◽

2018 ◽

Vol 5 (6) ◽

pp. 667-672 ◽

Cited By ~ 6

Author(s):

Inokentijs Josts ◽

Stephan Niebling ◽

Yunyun Gao ◽

Matteo Levantino ◽

Henning Tidow ◽

...

Keyword(s):

Small Molecule ◽

Conformational Changes ◽

Structural Changes ◽

Time Resolved ◽

Protein Dimerization ◽

X Ray ◽

Single Turnover ◽

X Ray Scattering ◽

Photoactivatable Proteins ◽

Ray Scattering

This work demonstrates a new method for investigating time-resolved structural changes in protein conformation and oligomerization via photocage-initiated time-resolved X-ray solution scattering by observing the ATP-driven dimerization of the MsbA nucleotide-binding domain. Photocaged small molecules allow the observation of single-turnover reactions of non-naturally photoactivatable proteins. The kinetics of the reaction can be derived from changes in X-ray scattering associated with ATP-binding and subsequent dimerization. This method can be expanded to any small-molecule-driven protein reaction with conformational changes traceable by X-ray scattering where the small molecule can be photocaged.

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2P031 1G1505 Conformational changes upon chloride-ion pumping during the pharaonis halorhodopsin photocycle studied by time-resolved FTIR spectroscopy(The 48th Annual Meeting of the Biophysical Society of Japan)

Seibutsu Butsuri ◽

10.2142/biophys.50.s87_3 ◽

2010 ◽

Vol 50 (supplement2) ◽

pp. S87

Author(s):

Tetsunari Kimura ◽

Hideki Kandori ◽

Yuji Furutani

Keyword(s):

Ftir Spectroscopy ◽

Annual Meeting ◽

Conformational Changes ◽

Chloride Ion ◽

Time Resolved ◽

Biophysical Society ◽

Ion Pumping

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Time-Resolved X-ray Absorption Determination of Structural Changes following Photoinduced Electron Transfer within Bis-porphyrin Heme Protein Models

The Journal of Physical Chemistry B ◽

10.1021/jp990056j ◽

1999 ◽

Vol 103 (16) ◽

pp. 3270-3274 ◽

Cited By ~ 20

Author(s):

Lin X. Chen ◽

Peter L. Lee ◽

David Gosztola ◽

Walter A. Svec ◽

Pedro A. Montano ◽

...

Keyword(s):

Electron Transfer ◽

Photoinduced Electron Transfer ◽

Structural Changes ◽

Heme Protein ◽

Time Resolved ◽

X Ray ◽

Absorption Determination ◽

Protein Models ◽

X Ray Absorption

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The kinetics of conformational changes of α2 -macroglobulin determined by time resolved X-ray solution scattering

FEBS Letters ◽

10.1016/0014-5793(94)80267-x ◽

1994 ◽

Vol 337 (2) ◽

pp. 171-174 ◽

Cited By ~ 5

Author(s):

Hideo Arakawa ◽

Takuji Urisaka ◽

Hirotsugu Tsuruta ◽

Yoshiyuki Amemiya ◽

Hiroshi Kihara ◽

...

Keyword(s):

Conformational Changes ◽

Time Resolved ◽

X Ray ◽

Α2 Macroglobulin ◽

Kinetics Of

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Predicting data quality in biological X-ray solution scattering

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318005004 ◽

2018 ◽

Vol 74 (8) ◽

pp. 727-738

Author(s):

Chenzheng Wang ◽

Yuexia Lin ◽

Devin Bougie ◽

Richard E. Gillilan

Keyword(s):

Conformational Changes ◽

Sample Path ◽

Flow Time ◽

Photon Flux ◽

Detector Efficiency ◽

Time Resolved ◽

Short Sample ◽

X Ray ◽

Path Lengths ◽

Flux Energy

Biological small-angle X-ray solution scattering (BioSAXS) is now widely used to gain information on biomolecules in the solution state. Often, however, it is not obvious in advance whether a particular sample will scatter strongly enough to give useful data to draw conclusions under practically achievable solution conditions. Conformational changes that appear to be large may not always produce scattering curves that are distinguishable from each other at realistic concentrations and exposure times. Emerging technologies such as time-resolved SAXS (TR-SAXS) pose additional challenges owing to small beams and short sample path lengths. Beamline optics vary in brilliance and degree of background scatter, and major upgrades and improvements to sources promise to expand the reach of these methods. Computations are developed to estimate BioSAXS sample intensity at a more detailed level than previous approaches, taking into account flux, energy, sample thickness, window material, instrumental background, detector efficiency, solution conditions and other parameters. The results are validated with calibrated experiments using standard proteins on four different beamlines with various fluxes, energies and configurations. The ability of BioSAXS to statistically distinguish a variety of conformational movements under continuous-flow time-resolved conditions is then computed on a set of matched structure pairs drawn from the Database of Macromolecular Motions (http://molmovdb.org). The feasibility of experiments is ranked according to sample consumption, a quantity that varies by over two orders of magnitude for the set of structures. In addition to photon flux, the calculations suggest that window scattering and choice of wavelength are also important factors given the short sample path lengths common in such setups.

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

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314092249 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C775-C775 ◽

Cited By ~ 1

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.

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