24 research outputs found
Crystallography-assisted Raman spectroscopy: X-ray induced photodissociation monitored by Raman microscopy
No full textCurrently, few sites provide the possibility to perform simultaneous Raman/X-ray diffraction of single macromolecular crystals. In this recent Raman-assisted crystallography applications, Raman microscopy has been representing a fine servant of a dominant X-ray Crystallography, and its highest scope was the reinforcement of structural data [1, 2]. The strategy of our work is to overturn this paradigm, focusing Crystallography-assisted Raman on the rich spectroscopic information that are well transferable to many bio-analytical applications. A Synchrotron with available Raman microscope, SLS [3], has been used to collect high-resolution crystallographic data on unusual states of model biomolecules. Two cases of study are presented: a) NO photodissociation in hemoglobin crystal structure; b) ultra high resolution crystal structure (0.8 Å) of ribonuclease A at different X- ray doses.
[1] Katona, G., et al. (2007) Science, 316, 449. [2] Vergara, A et al. (2010) J. Biol. Chem. 285,, 32568. [3] Owen, R. L., et al. (2009). J. Synchrotron Rad. 16, 173-182
Conformational transitions driven by PLP uptake in the psychrophilic serine hydroxymethyltransferase from Psychromonas ingrahamii
No full textSerine hydroxymethyltransferase (SHMT) is a pyridoxal-5’-phosphate (PLP)-dependent enzyme belonging to the fold type I superfamily which catalyzes in vivo the reversible conversion of l-serine and tetrahydropteroylglutamate (H4PteGlu) to glycine and 5,10-methylenetetrahydropteroylglutamate (5,10-CH2-H4PteGlu). The SHMT from the psychrophilic bacterium Psychromonas ingrahamii (piSHMT) had been recently purified and characterized. This enzyme was shown to display catalytic and stability properties typical of psychrophilic enzymes, namely high catalytic activity at low temperature and thermolability. To gain deeper insights into the structure-function relationship of piSHMT, the three-dimensional structure of its apo form was determined by X-ray crystallography. Homology modelling techniques were applied to build a model of the piSHMT holo form. Comparison of the two forms unraveled the conformation modifications that take place when the apo enzyme binds its cofactor. Our results show that the apo form is in an “open” conformation and possesses four (or five, in chain A) disordered loops whose electron density is not visible by X-ray crystallography. These loops contain residues that interact with the PLP cofactor and three of them are localized in the major domain that, along with the small domain, constitutes the single subunit of the SHMT homodimer. Cofactor binding triggers a rearrangement of the small domain that moves toward the large domain and screens the PLP binding site at the solvent side. Comparison to the mesophilic apo SHMT from Salmonella typhimurium suggests that the backbone conformational changes are wider in psychrophilic SHMT. © Proteins 2014;. © 2014 Wiley Periodicals, Inc
Selective X-ray induced NO-photodissociation in hemoglobin crystals: evidences from a Raman assisted-crystallographic study
No full textDespite the high physiological relevance, hemoglobin crystal structures with NO bound to heme constitute less than 1 % of the total ligated hemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO ligated Hbs is most likely related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O2. Here, using Raman assisted-X-ray crystallography, we show that under X-ray exposure (at four different radiation doses), crystals of nitrosylated hemoglobin from Trematomus bernacchii undergo a transition, mainly at the β chains, generating a pentacoordinate species, due to photodissociation of the Fe-NO bond. These data give a physical explanation of the low content of nitrosylated Hb structures available in the literature and provide a rough estimate of the relative Raman cross-section of bands corresponding to deoxygenated and nitrosylated hemes
Selective X-ray induced NO-photodissociation in hemoglobin crystals: evidences from a crystallography-assisted Raman microscopy study
No full textX-ray induced radiation damage is a frequent phenomenon, especially when using third generation synchrotrons. Raman microscopy prior and after X-ray data collection can be a valuable tool to detect artifacts derived from radiation damage. Here we propose X-ray photolysis on crystals of NO-Hb as an additional experimental source of denitrosylation. The system under investigation is the Hb from an Antarctic fish, Trematomus bernacchii (HbTb). To the best of our knowledge, this is the first Raman-assisted crystallographic evidence of X-ray induced NO-photodissociation at a 3rd generation synchrotron (beamline X10SA of the Swiss Light Source)
A combined Raman microspectroscopic / crystallographic approach to photolytic and oxidative denitrosylation in hemoglobins
No full textDespite the high physiological relevance, hemoglobin crystal structures with NO bound to heme are less than 1 % of the total ligated hemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO ligated Hbs is probably related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O2. We investigated the oxidative denitrosylation of the hemoglobin from Trematomus bernacchii (HbTb) via Raman-assisted crystallography. Furthermore, using X-ray crystallography-assisted Raman microscopy at the Swiss Light Source [1], we show that under X-ray exposure (at four different radiation doses), crystals of nitrosylated HbTb undergo a selective transition at the β chains generating a pentacoordinate species, due to photodissociation of the Fe-NO bond. This phenomenon was recently observed also in HbA solutions [2]. These observations give a physical explanation of the low content of nitrosylated Hb structures available in the literature and provide a valuable quantitative estimate of the relative Raman cross-section of bands corresponding to deoxygenated and nitrosylated hemes. The assembly of structures at different nitrosylation states provides a wide snapshot of the communication mechanism between α and β subunits of deoxygenated HbTb upon undergoing nitrosylation[3]. PNRA is acknowledged for financial support
Crystal structure of the ternary FimC-FimF(t)-FimD(N) complex indicates conserved pilus chaperone-subunit complex recognition by the usher FimD
No full textType 1 pili, anchored to the outer membrane protein FimD, enable uropathogenic Escherichia coli to attach to host cells. During pilus biogenesis, the N-terminal periplasmic domain of FimD (FimD(N)) binds complexes between the chaperone FimC and pilus subunits via its partly disordered N-terminal segment, as recently shown for the FimC-FimH(P)-FimD(N) ternary complex. We report the structure of a new ternary complex (FimC-FimF(t)-FimD(N)) with the subunit FimF(t) instead of FimH(p). FimD(N) recognizes FimC-FimF(t) and FimC-FimH(P) very similarly, predominantly through hydrophobic interactions. The conserved binding mode at a "hot spot" on the chaperone surface could guide the design of pilus assembly inhibitors
Structural Insights Into the Opening Mechanism of C1C2 Channelrhodopsin
No full textChannelrhodopsins, light-gated cation channels, enable precise control of neural cell depolarization or hyperpolarization with light in the field of optogenetics. This study integrates time-resolved serial crystallography and atomistic molecular dynamics (MD) simulations to resolve the structural changes during C1C2 channelrhodopsin activation. Our observations reveal that within the crystal environment, C1C2 predominantly remains in a light-activated state with characteristics of the M390 intermediate. Here, rearrangement of retinal within its binding pocket partially opens the central gate toward the extracellular vestibule. These structural changes initiate channel opening but were insufficient to allow K+ flow. Adjusting protonation states to represent the subsequent N520 intermediate in our MD simulations induced further conformational changes, including rearrangements of transmembrane helices 2 and 7, that opened the inner gate and the putative ion-translocation pathway. This allowed spontaneous cation conduction with low conductance, aligning with experimental findings. Our findings provide critical structural insights into key intermediates of the channel opening mechanism, enhancing our understanding of ion conduction and selectivity in channelrhodopsins at an atomistic level.European Research Council 10.13039/501100000781Deutsche Forschungsgemeinschaft 10.13039/501100001659Deutsche Forschungsgemeinschaft 10.13039/501100001659Deutsche Forschungsgemeinschaft 10.13039/501100001659Schweizerischer Nationalfonds zur F?rderung der Wissenschaftlichen Forschung 10.13039/501100001711Schweizerischer Nationalfonds zur F?rderung der Wissenschaftlichen Forschung 10.13039/501100001711Gemeinn?tzige Hertie-Stiftung 10.13039/501100003493Innosuisse - Schweizerische Agentur f?r Innovationsf?rderung 10.13039/501100013348Peer Reviewe
Time-resolved X-ray diffraction and solution scattering studies of Sensory Rhodopsin II in isolation and in complex with its transducer
No full textLight is an important source of energy for many living organisms.
Many life forms have therefore evolved cellular receptors that are able to
sense light and thereby optimise conditions for photosynthesis and pho-
totrophy. Microbial Rhodopsins are a family of heptahelical transmem-
brane proteins characterised by the presence of a retinal chromophore
bound to a conserved lysine of helix seven. When the retinal absorbs a
photon, it photoisomerises from an all-trans to a 13-cis conformation.
Sensory Rhodopsin II (SRII) is a microbial rhodopsin identified in the
halophilic archaeon Nantronomonas pharaonis. Together with its trans-
ducer protein HtrII, SRII it initiates a photophobic reaction of the host
in response to blue light. Conformational changes within this complex
are sensed by the HAMP domain of HtrII and trigger a signalling cas-
cade controlled by the so-called two-component system (TCS). The TCS
is ubiquitous in prokaryotes and is present in some eukaryotes. This im-
plies significant pharmaceutical interest due to the involvement of TCS
in bacterial virulence, antibiotic resistance, and phototaxis. Many details
concerning the mechanisms of signal transduction through the SRII:HtrII
complex remain unclear. In this work I aimed to address these questions by
observing the nature and extent of secondary structural rearrangements
in SRII in isolation and in complex with HtrII using time-resolved serial
synchrotron X-ray crystallography (TR-SSX) and time-resolved X-ray so-
lution scattering (TR-XSS). In PAPER I, we collected room-temperature
TR-XSS data on SRII in isolation and compared the observed structural
changes with those observed in bacteriorhodopsin (bR), a heavily studied
light-driven proton pump. Our observations provide structural insight into
why these very similar proteins have very different photocycle duration.
In both proteins, helix F undergoes an outward movement, yet structural
rearrangement within helix G are suppressed in SRII, resulting in a slower
photocycle and reflecting its function as a signalling receptor. In PAPER II
we observed the structure of the SRII:HtrII complex at room-temperature
using serial synchrotron x-ray crystallography (SSX). Our data provides the first room-temperature structure of the SRII:HtrII complex and al-
lows five additional residues to be modelled on the cytoplasmic side of
transmembrane helix 1 (TM1) of HtrII. In Paper III we used TR-SSX to
investigate light-initiated conformational changes of within the SRII:HtrII
complex. Our observations show how a structural signal originating at the
retinal is transferred from SRII to HtrII. A preliminary structural analysis
suggests that an outward movement of helix F of SRII is translated into
a piston-like movement of transmembrane helix 2 (TM2) towards the cy-
toplasm, a model that is largely consistent with the conclusions of earlier
cryo-trapping studies. In Paper IV we used TR-XSS to analyse conforma-
tional changes in SRII and the SRII:HtrII complex. As a solution phase
method, TR-XSS is complementary to crystallography and protein mo-
tions are not constrained by a crystal lattice, but the information content
is lower. Our TR-XSS data were consistent with a light-induced outward
movement of the cytoplasmic portions of helices E and F, and more subtle
movements in helices C, D and E. Structural rearrangements in helices E
and F are less extensive when the transducer binds to SRII. These results
increase our understanding of how a light signal is sensed by the photo-
taxis receptor SRII, and how this signal is transmitted to its transducer
protein, HtrII
Testing the limits: serial crystallography using unpatterned fixed targets
No full textSheet-on-sheet (SOS) fixed-target chips are arguably the most versatile, cheapest and simplest sample-delivery method for ambient-temperature data acquisition using serial crystallography approaches at synchrotrons and X-ray free-electron lasers (XFELs). Their defining feature, the absence of any hard-patterned restrictions around crystals, is their strength as it removes limitations on crystal sizes or environments. However, it is also their weakness when it comes to limiting undesired effects on yet-to-be-irradiated crystals due to diffusing heat, radicals or gas originating from previous exposures. We explored whether SOS chips can be used for damage-free serial data collection on the new ID29 beamline at the ESRF-EBS, a fourth-generation synchrotron light source, as well as at the new Cristallina-MX station at SwissFEL. We collected serial data sets from microcrystals of the hemoprotein DtpAa, which was reported to have a highly radiation-sensitive iron–water bond length. The data sets differ in step size between exposures within and between lines of a serpentine-like data-acquisition scan. We observe no significant changes in the distance of the water ligand of the heme in the structures obtained from the ID29 SSX data. However, when compared with those collected at Cristallina-MX, the diffraction intensities collected at ID29 suggest global damage akin to Bragg termination occurring during the 90 µs exposure at ID29. Moreover, differences in the heme geometry and the proximal histidine–iron bond length point to local damage in all ID29 data sets regardless of the X-ray spacing. SFX data collected at Cristallina-MX show a phase transition of the DtpAa crystal lattice for X-ray step sizes of ≤20 µm. This phase transition might be caused by heating and/or hydrogen-gas-induced crystal dehydration. Vigilance remains required to safeguard against radiation damage at fourth-generation synchrotrons and XFELs
Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography
Get PDFThe binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we capture the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.Photopharmacology manipulates the biological activity of small molecules by light. Using an X-ray laser, the authors follow the release of the drug azo-combretastatin A4 from tubulin and the concomitant structural changes over nine orders of magnitude in time
