scholarly journals Unidirectional ion transport mechanism of a light-driven chloride pump revealed using X-ray free electron lasers

2021 ◽  
Author(s):  
Toshiaki Hosaka ◽  
Takashi Nomura ◽  
Minoru Kubo ◽  
Takanori Nakane ◽  
Luo Fangjia ◽  
...  
Keyword(s):  
Ion Transport ◽  
Conformational Changes ◽  
Structural Changes ◽  
Ion Transfer ◽  
Ion Release ◽  
Time Resolved ◽  
Iodide Ions ◽  
Lateral Displacements ◽  
Extracellular Side ◽  
Cytoplasmic Side

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 µs and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

scholarly journals Structural photoactivation of a full-length bacterial phytochrome

2016 ◽  
Vol 2 (8) ◽  
pp. e1600920 ◽  
Author(s):  
Alexander Björling ◽  
Oskar Berntsson ◽  
Heli Lehtivuori ◽  
Heikki Takala ◽  
Ashley J. Hughes ◽  
...  
Keyword(s):  
Time Scales ◽  
Conformational Changes ◽  
Structural Changes ◽  
Deinococcus Radiodurans ◽  
Full Length ◽  
Photon Absorption ◽  
Signaling Mechanism ◽  
Time Resolved ◽  
Binding Domains ◽  
Sensor Proteins

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.

scholarly journals Retinal isomerization and water-pore formation in channelrhodopsin-2

2018 ◽  
Vol 115 (14) ◽  
pp. 3557-3562 ◽  
Author(s):  
Albert Ardevol ◽  
Gerhard Hummer
Keyword(s):  
Schiff Base ◽  
Conformational Changes ◽  
Pore Formation ◽  
Proton Acceptor ◽  
Time Resolved ◽  
Channel Opening ◽  
Selective Channel ◽  
Extracellular Side ◽  
Membrane Spanning ◽  
Time Resolved Infrared Spectroscopy

Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel widely used in optogenetics. Photoactivation triggers a trans-to-cis isomerization of a covalently bound retinal. Ensuing conformational changes open a cation-selective channel. We explore the structural dynamics in the early photocycle leading to channel opening by classical (MM) and quantum mechanical (QM) molecular simulations. With QM/MM simulations, we generated a protein-adapted force field for the retinal chromophore, which we validated against absorption spectra. In a 4-µs MM simulation of a dark-adapted ChR2 dimer, water entered the vestibules of the closed channel. Retinal all-trans to 13-cis isomerization, simulated with metadynamics, triggered a major restructuring of the charge cluster forming the channel gate. On a microsecond time scale, water penetrated the gate to form a membrane-spanning preopen pore between helices H1, H2, H3, and H7. This influx of water into an ion-impermeable preopen pore is consistent with time-resolved infrared spectroscopy and electrophysiology experiments. In the retinal 13-cis state, D253 emerged as the proton acceptor of the Schiff base. Upon proton transfer from the Schiff base to D253, modeled by QM/MM simulations, we obtained an early-M/P2390–like intermediate. Rapid rotation of the unprotonated Schiff base toward the cytosolic side effectively prevents its reprotonation from the extracellular side. From MM and QM simulations, we gained detailed insight into the mechanism of ChR2 photoactivation and early events in pore formation. By rearranging the network of charges and hydrogen bonds forming the gate, water emerges as a key player in light-driven ChR2 channel opening.

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

2017 ◽  
Vol 114 (52) ◽  
pp. 13708-13713 ◽  
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.

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

IUCrJ ◽  
2018 ◽  
Vol 5 (6) ◽  
pp. 667-672 ◽  
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.

scholarly journals Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kazumasa Oda ◽  
Takashi Nomura ◽  
Takanori Nakane ◽  
Keitaro Yamashita ◽  
Keiichi Inoue ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Local Deformation ◽  
Proton Donor ◽  
Time Resolved ◽  
Molecular Events ◽  
Channel Opening ◽  
Ion Conducting ◽  
And Shifts ◽  
Opening And Closing

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

scholarly journals Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions

2019 ◽  
Vol 5 (7) ◽  
pp. eaaw1531 ◽  
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.

scholarly journals Crystallisation and conformation-controlled breathing of the Al-MOF CAU-13

2014 ◽  
Vol 70 (a1) ◽  
pp. C62-C62
Author(s):  
Alexandra Lieb ◽  
Felicitas Niekiel ◽  
Norbert Stock
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Metal Organic Frameworks ◽  
Structural Topology ◽  
Time Resolved ◽  
Metal Organic ◽  
Synchotron Radiation ◽  
Induction Times ◽  
Linker Molecules

Within the last couple of years an enormous variety of metal-organic frameworks (MOFs) has been synthesized. Besides characteristics like large specific surface area or a defined interaction with gas molecules a "breathing" effect was described as well. During this breathing the atoms can undergo certain movements keeping the same structural topology and without losing crystallinity. Most known MOFs contain rigid aromatic linker molecules. A rare example of a MOF with flexible aliphatic linkers is [Al(OH)(trans CDC)] (CDC = cyclohexanedicarboxylate), known as CAU 13.[1] The structure was determined from PXRD-data using synchotron radiation. In analogy to Al-MIL 53,[2] CAU 13 is build up from chains of corner-sharing AlO6-octahedra interconnected by the linker molecules. Interestingly, the trans-CDC ion is incorporated in a,a- as well as e,e-conformation. Time-resolved in-situ EDXRD experiments at HASYLAB (DESY, Hamburg) show short induction times for the crystallization of CAU-13. Full crystallization occurs within two hours even at low reaction temperatures. CAU-13[3] shows porosity towards different adsoptives after thermal activation. In-situ temperature dependent PXRD experiments show a widening of the pores along the b-axis up to 3500C. The flexibility of the linker molecules allows structural changes of the compound during adsorption. While the adsorption of hydrophilic molecules only cause a small breathing effect, the adsorption of xylene leads to drastic changes in the crystal structure. The a,a-CDC2- ions change conformation to e,e-type to increase the cell volume per formula sum by 25 %. This combination of pore widening and conformational changes constitutes a new type of breathing in Metal-Organic Frameworks.

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

2021 ◽  
Vol 118 (13) ◽  
pp. e2020486118
Author(s):  
Ji-Hye Yun ◽  
Xuanxuan Li ◽  
Jianing Yue ◽  
Jae-Hyun Park ◽  
Zeyu Jin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Early Stage ◽  
Chloride Ion ◽  
Time Resolved ◽  
X Ray ◽  
Combining Data ◽  
Linac Coherent Light Source ◽  
Dynamics Simulations ◽  
Ion Pumping

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.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

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