| 11. |
- Malmerberg, Erik, 1980, et al.
(author)
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Conformational activation of visual rhodopsin in native disc membranes
- 2015
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In: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 8:367
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Journal article (peer-reviewed)abstract
- Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested.
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| 12. |
- Marcellini, Moreno, et al.
(author)
-
Transient isomers in the photodissociation of bromoiodomethane
- 2018
-
In: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 148:13
-
Journal article (peer-reviewed)abstract
- The photochemistry of halomethanes is fascinating for the complex cascade reactions toward either the parent or newly synthesized molecules. Here, we address the structural rearrangement of photodissociated CH2IBr in methanol and cyclohexane, probed by time-resolved X-ray scattering in liquid solution. Upon selective laser cleavage of the C-I bond, we follow the reaction cascade of the two geminate geometrical isomers, CH2I-Br and CH2Br-I. Both meta-stable isomers decay on different time scales, mediated by solvent interaction, toward the original parent molecule. We observe the internal rearrangement of CH2Br-I to CH2I-Br in cyclohexane by extending the time window up to 3 mu s. We track the photoproduct kinetics of CH2Br-I in methanol solution where only one isomer is observed. The effect of the polarity of solvent on the geminate recombination pathways is discussed. Published by AIP Publishing.
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| 13. |
- Nango, E., et al.
(author)
-
A three-dimensional movie of structural changes in bacteriorhodopsin
- 2016
-
In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 354:6319, s. 1552-1557
-
Journal article (peer-reviewed)abstract
- Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.
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| 14. |
- Neutze, Richard, 1969, et al.
(author)
-
Membrane protein structural biology using X-ray free electron lasers
- 2015
-
In: Current Opinion in Structural Biology. - : Elsevier BV. - 0959-440X .- 1879-033X. ; 33, s. 115-125
-
Journal article (peer-reviewed)abstract
- © 2015 . Membrane protein structural biology has benefitted tremendously from access to micro-focus crystallography at synchrotron radiation sources. X-ray free electron lasers (XFELs) are linear accelerator driven X-ray sources that deliver a jump in peak X-ray brilliance of nine orders of magnitude and represent a disruptive technology with potential to dramatically change the field. Membrane proteins were amongst the first macromolecules to be studied with XFEL radiation and include proof-of-principle demonstrations of serial femtosecond crystallography (SFX), the observation that XFEL data can deliver damage free crystallographic structures, initial experiments towards recording structural information from 2D arrays of membrane proteins, and time-resolved SFX, time-resolved wide angle X-ray scattering and time-resolved X-ray emission spectroscopy studies. Conversely, serial crystallography methods are now being applied using synchrotron radiation. We believe that a context dependent choice of synchrotron or XFEL radiation will accelerate progress towards novel insights in understanding membrane protein structure and dynamics.
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| 15. |
- Nogly, P., et al.
(author)
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Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography
- 2016
-
In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
-
Journal article (peer-reviewed)abstract
- Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 angstrom resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
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| 16. |
- Nogly, P., et al.
(author)
-
Lipidic cubic phase serial millisecond crystallography using synchrotron radiation
- 2015
-
In: Iucrj. - : International Union of Crystallography (IUCr). - 2052-2525. ; 2
-
Journal article (peer-reviewed)abstract
- Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 angstrom. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.
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| 17. |
- Nogly, P., et al.
(author)
-
Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser
- 2018
-
In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 361:6398
-
Journal article (peer-reviewed)abstract
- INTRODUCTION Retinal is a light-sensitive protein ligand that is used by all domains of life to process the information and energy content of light. Retinal-binding proteins are integral membrane proteins that drive vital biological processes, including light sensing for spatial orientation and circadian clock adjustment, as well as maintaining electrochemical gradients through ion transport. They also form the basis for optogenetic manipulation of neural cells. How the protein environment guides retinal isomerization on a subpicosecond time scale toward a single high-yield product is a fundamental outstanding question in photobiology. RATIONALE Light-induced isomerization of retinal is among the fastest reactions known in biology. It has been widely studied by spectroscopic techniques to probe the evolution of spectral intermediates over time. Using x-ray free-electron lasers (XFELs), it is now possible to observe ultrafast photochemical reactions and their induced molecular motions within proteins on scales of femtoseconds to milliseconds with near-atomic structural resolution. In this work, we used XFEL radiation to study the structural dynamics of retinal isomerization in the light-driven proton-pump bacteriorhodopsin (bR). The principal mechanism of isomerization in this prototypical retinal-binding protein has direct relevance for all other members of this important family of membrane proteins, and it provides insight into how protein environments catalyze photochemical reactions in general. RESULTS We collected high-resolution x-ray diffraction data from bR microcrystals injected across the femtosecond x-ray pulses of the Linac Coherent Light Source after excitation of the retinal chromophore by an optical laser pulse. X-ray diffraction images were sorted into temporal subgroups with a precision of about 200 fs. A series of 18 overlapping difference Fourier electron density maps reveal structural changes over the first picosecond of retinal photoexcitation. Complementary data for time delays of 10 ps and 8.33 ms allow us to resolve the later stages of the reaction. In combination with refined crystallographic structures at pump-probe delays corresponding to where the spectroscopically characterized I, J, K, and M intermediates form in solution, our time-resolved structural data reveal the trajectory of retinal isomerization and provide atomic details at key points along the reaction. The aspartic acid residues of the retinal counterion and functional water molecules in close proximity to the retinal Schiff base respond collectively to the formation and decay of the excited state. This collective motion sets the stage for retinal isomerization, which proceeds via a twisted retinal configuration. Quantum mechanics/molecular mechanics simulations provide theoretical support for this structural evolution. CONCLUSION Our observations reveal how, concomitant with the formation of the earliest excited state, the retinal-binding pocket opens up in close proximity to the isomerizing bond. We propose that ultrafast charge transfer along retinal is a driving force for collective motions that contribute to the stereoselectivity and efficiency of retinal isomerization within a protein scaffold. Vibrational quake-like motions extending from retinal to the protein may also be a mechanism through which excess energy is released in a nonradiative fashion.
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| 18. |
- Sharma, Amit, et al.
(author)
-
A simple adaptation to a protein crystallography station to facilitate difference X-ray scattering studies
- 2019
-
In: Journal of Applied Crystallography. - : International Union of Crystallography (IUCr). - 0021-8898 .- 1600-5767. ; 52, s. 378-386
-
Journal article (peer-reviewed)abstract
- The X-ray crystallography station I911-2 at MAXLab II (Lund, Sweden) has been adapted to enable difference small- and wide-angle X-ray scattering (SAXS/WAXS) data to be recorded. Modifications to the beamline included a customized flow cell, a motorized flow cell holder, a helium cone, a beam stop, a sample stage and a sample delivery system. This setup incorporated external devices such as infrared lasers, LEDs and reaction mixers to induce conformational changes in macromolecules. This platform was evaluated through proof-of-principle experiments capturing light-induced conformational changes in phytochromes. A difference WAXS signature of conformational changes in a plant aquaporin was also demonstrated using caged calcium.
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| 19. |
- Sharma, Amit, et al.
(author)
-
A simple adaptation to a protein crystallography station to facilitate difference X-ray scattering studies
- 2019
-
In: Journal of Applied Crystallography. - 1600-5767. ; 52:2, s. 378-386
-
Journal article (peer-reviewed)abstract
- The X-ray crystallography station I911-2 at MAXLab II (Lund, Sweden) has been adapted to enable difference small- and wide-angle X-ray scattering (SAXS/WAXS) data to be recorded. Modifications to the beamline included a customized flow cell, a motorized flow cell holder, a helium cone, a beam stop, a sample stage and a sample delivery system. This setup incorporated external devices such as infrared lasers, LEDs and reaction mixers to induce conformational changes in macromolecules. This platform was evaluated through proof-of-principle experiments capturing light-induced conformational changes in phytochromes. A difference WAXS signature of conformational changes in a plant aquaporin was also demonstrated using caged calcium.
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| 20. |
- Sharma, Amit, et al.
(author)
-
Asymmetry in serial femtosecond crystallography data
- 2017
-
In: Acta Crystallographica a-Foundation and Advances. - : International Union of Crystallography (IUCr). - 2053-2733. ; 73, s. 93-101
-
Journal article (peer-reviewed)abstract
- Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background.
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