I24-Microfocus Macromolecular Crystallography
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James
Baxter
,
Christopher D. M.
Hutchison
,
Karim
Maghlaoui
,
Violeta
Cordon-Preciado
,
R. Marc L.
Morgan
,
Pierre
Aller
,
Agata
Butryn
,
Danny
Axford
,
Sam
Horrell
,
Robin L.
Owen
,
Selina L. S.
Storm
,
Nicholas E.
Devenish
,
Jasper J.
Van Thor
Diamond Proposal Number(s):
[17221]
Open Access
Abstract: The chromophores of reversibly switchable fluorescent proteins (rsFPs) undergo photoisomerization of both the trans and cis forms. Concurrent with cis/trans photoisomerisation, rsFPs typically become protonated on the phenolic oxygen resulting in a blue shift of the absorption. A synthetic rsFP referred to as rsEospa, derived from EosFP family, displays the same spectroscopic behavior as the GFP-like rsFP Dronpa at pH 8.4 and involves the photoconversion between nonfluorescent neutral and fluorescent anionic chromophore states. Millisecond time-resolved synchrotron serial crystallography of rsEospa at pH 8.4 shows that photoisomerization is accompanied by rearrangements of the same three residues as seen in Dronpa. However, at pH 5.5 we observe that the OFF state is identified as the cationic chromophore with additional protonation of the imidazolinone nitrogen which is concurrent with a newly formed hydrogen bond with the Glu212 carboxylate side chain. FTIR spectroscopy resolves the characteristic up-shifted carbonyl stretching frequency at 1713 cm–1 for the cationic species. Electronic spectroscopy furthermore distinguishes the cationic absorption band at 397 nm from the neutral species at pH 8.4 seen at 387 nm. The observation of photoisomerization of the cationic chromophore state demonstrates the conical intersection for the electronic configuration, where previously fluorescence was proposed to be the main decay route for states containing imidazolinone nitrogen protonation. We present the full time-resolved room-temperature X-ray crystallographic, FTIR, and UV/vis assignment and photoconversion modeling of rsEospa.
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Nov 2022
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I15-1-X-ray Pair Distribution Function (XPDF)
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Diamond Proposal Number(s):
[27222]
Abstract: Water-in-salt systems, i.e., super-concentrated aqueous electrolytes, such as lithium bis(trifluoromethanesulfonyl)imide (21 mol/kgwater), have been recently discovered to exhibit unexpectedly large electrochemical windows and high lithium transference numbers, thus paving the way to safe and sustainable charge storage devices. The peculiar transport features in these electrolytes are influenced by their intrinsically nanoseparated morphology, stemming from the anion hydrophobic nature and manifesting as nanosegregation between anions and water domains. The underlying mechanism behind this structure–dynamics correlation is, however, still a matter of strong debate. Here, we enhance the apolar nature of the anions, exploring the properties of the aqueous electrolytes of lithium salts with a strongly asymmetric anion, namely, (trifluoromethylsulfonyl)(nonafluorobutylsulfonyl) imide. Using a synergy of experimental and computational tools, we detect a remarkable level of structural heterogeneity at a mesoscopic level between anion-rich and water-rich domains. Such a ubiquitous sponge-like, bicontinuous morphology develops across the whole concentration range, evolving from large fluorinated globules at high dilution to a percolating fluorous matrix intercalated by water nanowires at super-concentrated regimes. Even at extremely concentrated conditions, a large population of fully hydrated lithium ions, with no anion coordination, is detected. One can then derive that the concomitant coexistence of (i) a mesoscopically segregated structure and (ii) fully hydrated lithium clusters disentangled from anion coordination enables the peculiar lithium diffusion features that characterize water-in-salt systems.
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Nov 2021
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[20249]
Abstract: A detailed understanding of the local dynamics in ionic liquids remains an important aspect in the design of new ionic liquids as advanced functional fluids. Here, we use small-angle X-ray scattering and quasi-elastic neutron spectroscopy to investigate the local structure and dynamics in a model ionic liquid as a function of temperature and pressure, with a particular focus on state points (P,T) where the macroscopic dynamics, i.e., conductivity, is the same. Our results suggest that the initial step of ion transport is a confined diffusion process, on the nanosecond timescale, where the motion is restricted by a cage of nearest neighbors. This process is invariant considering timescale, geometry, and the participation ratio, at state points of constant conductivity, i.e., state points of isoconductivity. The connection to the nearest-neighbor structure is underlined by the invariance of the peak in the structure factor corresponding to nearest-neighbor correlations. At shorter timescales, picoseconds, two localized relaxation processes of the cation can be observed, which are not directly linked to ion transport. However, these processes also show invariance at isoconductivity. This points to that the overall energy landscape in ionic liquids responds in the same way to density changes and is mainly governed by the nearest-neighbor interactions.
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Mar 2021
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[23750]
Open Access
Abstract: Intrinsically disordered proteins (IDP) are proteins that sample a heterogeneous ensemble of conformers in solution. An estimated 25–30% of all eukaryotic proteins belong to this class. In vivo, IDPs function under conditions that are highly crowded by other biological macromolecules. Previous research has highlighted that the presence of crowding agents can influence the conformational ensemble sampled by IDPs, resulting in either compaction or expansion. The effects of self-crowding of the disordered protein Histatin 5 has, in an earlier study, been found to have limited influence on the conformational ensemble. In this study, it is examined whether the short chain length of Histatin 5 can explain the limited effects of crowding observed, by introducing (Histatin 5)2, a tandem repeat of Histatin 5. By utilizing small-angle X-ray scattering, it is shown that the conformational ensemble is conserved at high protein concentrations, in resemblance with Histatin 5, although with a lowered protein concentration at which aggregation arises. Under dilute conditions, atomistic molecular dynamics and coarse-grained Monte Carlo simulations, as well as an established scaling law, predicted more extended conformations than indicated by experimental data, hence implying that (Histatin 5)2 does not behave as a self-avoiding random walk.
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Dec 2020
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[19692]
Abstract: This study aimed at the development of improved drugs against human osteosarcoma, which is the most common primary bone tumor in children and teenagers with a low prognosis available treatment. New insights into the impact of an unconventional Pd(II) anticancer agent on human osteosarcoma cells were obtained by synchrotron-based infrared microspectroscopy (SR-microFTIR) and quasi-elastic neutron scattering (QENS) experiments from its effect on the cellular metabolism to its influence on intracellular water which can be regarded as a potential secondary pharmacological target. Specific infrared biomarkers of drug action were identified, enabling a molecular-level description of variations in cellular biochemistry upon drug exposure. The main changes were detected on the protein and lipid cellular components, namely on the ration of unsaturated-to-saturated fatty acids. QENS revealed a reduced water mobility within the cytoplasm for drug-treated cells, coupled to a disruption of the hydration layers of biomolecules. Additionally, the chemical and dynamical profiles of osteoscarcoma cells were compared to metastatic breast cancer, revealing distinct dissimilarities that may influence drug activity.
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Jul 2019
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[19477]
Open Access
Abstract: We investigate the impact of lipidation on the self-assembly of two peptide fragments from the gastrointestinal peptide hormone PYY3-36. The lipopeptides C16IKPEAP and C16IKPEAPGE contain the first 6 and 8 amino acid residues respectively from the PYY3-36 peptide sequence, with a palmitoyl C16 tail attached at the N-terminus. These lipopeptides form spherical micelles in aqueous solution, above a critical micelle concentration (cmc), which is pH-dependent. Modelling of small-angle x-ray scattering data along with molecular dynamics simulations shows the formation of micelles with a hydrophobic interior and a well hydrated exterior. The lipopeptides have a disordered conformation over the pH and temperature range studied. The cmc is found to be independent of temperature, pointing to athermal micellization. In contrast to the presence of hydrated micelles in solution, β-sheet amyloid fibrils form in dried samples. Thus, the nanostructure of lipidated PYY3-36 fragment peptides can be tuned by control of pH or concentration, for future applications.
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Jan 2019
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B23-Circular Dichroism
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Parveen
Akhtar
,
Dominik
Lindorfer
,
Monika
Lingvay
,
Krzysztof
Pawlak
,
Otto
Zsiros
,
Giuliano
Siligardi
,
Tamas
Javorfi
,
Márta
Dorogi
,
Bettina
Ughy
,
Gyozo
Garab
,
Thomas
Renger
,
Petar H.
Lambrev
Diamond Proposal Number(s):
[15094, 16232, 17698, 19120, 20644]
Abstract: Anisotropic circular dichroism (ACD) spectroscopy of macroscopically aligned molecules reveals additional information about their excited states that is lost in the CD of randomly-oriented solutions. ACD spectra of light-harvesting complex II (LHCII)—the main peripheral antenna of photosystem II in plants—in oriented lipid bilayers, were recorded from the far-UV to the visible wavelength region. The ACD spectra show a drastically enhanced magnitude and level of detail compared to the isotropic CD spectra, resolving a greater number of bands and weak optical transitions. Exciton calculations show that the spectral features in the chlorophyll Qy region are well reproduced by an existing Hamiltonian for LHCII, providing further evidence for the identity of energy sinks at chlorophylls a603 and a610 in the stromal layer and chlorophylls a604 and a613 in the luminal layer. We propose ACD spectroscopy to be a valuable tool linking the three-dimensional structure and the photophysical properties of pigment–protein complexes.
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Jan 2019
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[15990]
Abstract: The early stage crystallisation behaviour in a triacylglycerol mixture has been investigated on the nanoscale with a novel global small angle X-ray scattering analysis technique. This method has been tailored for the determination of the electron density profiles (EDP) replicating both, (i) the nanostructural texture of molten TAGs (refer to part I ‘Global Small-Angle X-ray Scattering Data Analysis of Triacylglycerols in the Molten State’ of this publication series), and (ii) the lamellar structure of the metastable α-polymorph. In a first stage, the α-phase scattering contribution alone was examined by classical Fourier analysis as well as by globally fitting the data, leading to practically identical EDPs. Based on these findings, we extended our analysis to the entire X-ray scattering contribution arising from molten TAGs and the solid α-phase fraction. Remarkably, the experimental and theoretical data agree very well, providing for the first time a detailed nanostructural understanding about the coexisting molecular assemblies. This allowed in turn, also to quantitatively determine the solid fat content (SFC) with X-ray scattering data. Our new theoretical approach for measurement of SFC is based on the global analysis of small angle scattering/diffraction patterns and the SFC-results are in good agreement with values obtained from other techniques such as NMR spectroscopy.
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Oct 2018
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[15990]
Abstract: The study of triacylglycerols (TAGs) in their molten state is of fundamental importance for a deeper understanding of the TAG crystallization processes, being highly relevant for both manufacturing and medical applications. Although different models have been proposed to explain the nanostructured nature of the fluid state of TAGs, none of them are fully satisfactory. In this paper, we propose a new model consisting of positionally uncorrelated lamellar TAG assemblies embedded in an isotropic medium that assist as prenucleating structures. This model was validated by applying a novel global fitting method, resulting in an excellent agreement with the small-angle X-ray scattering data. A deeper analysis of the scattering patterns at different temperatures, both in cooling and heating directions, allowed us further to detect the crystalline traces of TAGs even after heating to 40 °C and record, on cooling, the onset of crystallization at 30–25 °C. The application of the presented novel model not only explains the outstandingly structured fluid of molten TAGs, but also lays the basis for analyzing first the crystallization steps in greater detail, which is outlined in our follow-up paper “Global Small-Angle X-ray Scattering Data Analysis of Triacylglycerols in the α-Phase (Part II)”.
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Oct 2018
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I04-Macromolecular Crystallography
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Abstract: Folding of proteins to their functional conformation is paramount to life. Though 75% of the proteome consists of multidomain proteins, our knowledge of folding has been based primarily on studies conducted on single-domain and fast-folding proteins. Nonetheless, the complexity of folding landscapes exhibited by multidomain proteins has received increased scrutiny in recent years. We study the three-domain protein adenylate kinase from E. coli (AK), which has been shown to fold through a series of pathways involving several intermediate states. We use a protein design method to manipulate the folding landscape of AK, and single-molecule FRET spectroscopy to study the effects on the folding process. Mutations introduced in the NMP binding (NMPbind) domain of the protein are found to have unexpected effects on the folding landscape. Thus, while stabilizing mutations in the core of the NMPbind domain retain the main folding pathways of wild-type AK, a destabilizing mutation at the interface between the NMPbind and the CORE domains causes a significant repartition of the flux between the folding pathways. Our results demonstrate the outstanding plasticity of the folding landscape of AK and reveal how specific mutations in the primary structure are translated into changes in folding dynamics. The combination of methodologies introduced in this work should prove useful for deepening our understanding of the folding process of multidomain proteins.
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Sep 2018
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