I24-Microfocus Macromolecular Crystallography
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Abstract: Understanding the photoinduced dynamics of fluorescent proteins is essential for their applications in
bioimaging. Despite numerous studies on the ultrafast dynamics, the delayed response of these proteins,
which often results in population of kinetically trapped dark states of various origins, is largely unexplored.
Here, by using transient absorption spectroscopy spanning the time scale from picoseconds to seconds, we
reveal a hidden reactivity of the bright blue-light emitting protein mKalama1 previously thought to be inert.
This protein shows no excited-state proton transfer during its nanosecond excited-state lifetime; however,
its tyrosine-based chromophore undergoes deprotonation coupled to non-radiative electronic relaxation.
Such deprotonation causes distinct optical absorption changes in the broad UV-to-NIR spectral range
(ca. 300–800 nm); the disappearance of the transient absorption signal has a complex nature and spans the
whole microsecond-to-second time scale. The mechanisms underlying the relaxation kinetics are disclosed
based on the X-ray structural analysis of mKalama1 and the high-level electronic structure calculations of
proposed intermediates in the photocycle. We conclude that the non-radiative excited-state decay includes
two major branches: internal conversion coupled to intraprotein proton transfer, where a conserved residue
E222 serves as the proton acceptor; and ionization induced by two consecutive resonant absorption events,
followed by deprotonation of the chromophore radical cation to bulk solvent through a novel watermediated
proton-wire pathway. Our findings open up new perspectives on the dynamics of fluorescent
proteins as tracked by its optical transient absorption in the time domain extending up to seconds.
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May 2015
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I24-Microfocus Macromolecular Crystallography
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Abstract: The temperature dependence of hydride transfer from the substrate to the N5 of the FAD cofactor during the reductive half-reaction of Pleurotus eryngii aryl-alcohol oxidase (AAO) is assessed here. Kinetic isotope effects on both the pre-steady state reduction of the enzyme and its steady-state kinetics, with differently deuterated substrates, suggest an environmentally-coupled quantum-mechanical tunnelling process. Moreover, those kinetic data, along with the crystallographic structure of the enzyme in complex with a substrate analogue, indicate that AAO shows a pre-organized active site that would only require the approaching of the hydride donor and acceptor for the tunnelled transfer to take place. Modification of the enzyme's active-site architecture by replacement of Tyr92, a residue establishing hydrophobic interactions with the substrate analogue in the crystal structure, in the Y92F, Y92L and Y92W variants resulted in different temperature dependence patterns that indicated a role of this residue in modulating the transfer reaction.
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Oct 2017
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[6719]
Abstract: Polyhedral oligomeric silsesquioxane (POSS) cubic cage systems (octa-n-octadecyloctasilsesquioxane, (T8C18) and octakis(n-octadecyldimethylsiloxy)octasilsesquioxane, (Q8C18)) were synthesised with eight long n-alkyl chain (R = C18H37) substituent arms, as model nano-functionalized compounds. The crystalline packing morphology of the cages was studied using time-resolved Small- and Wide-angle X-ray scattering (SAXS/WAXS), thermal and optical techniques. From thermal analysis the melting and crystallization temperatures of the Q8 cage were significantly less than those for the T8 cage. X-ray scattering showed that both cage systems have long-range crystalline ordering where the alkyl chains align in a parallel axial disposition from the POSS core giving a ‘rod-like’ self-assembled packing morphology. The packing length-scale can be directly related to the overall dimensions of the POSS molecules. Compared to the T8 cages, the Q8 cages pack more efficiently allowing the interdigitation of the alkyl chain arms. Different packing modes and thermal behaviour observed for the T8 and Q8 cages is directly attributed to their structural chemistry. For the Q8 cage, the presence of the OSiMe2 spacer groups which tether the alkyl chain arms to the cage (absent in the T8 cages) allows greater flexibility of the arms letting them interdigitate with each other when packing which is not observed for the analogous T8 cages.
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Feb 2013
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[7976]
Abstract: We present a study on ion specific effects on the intermolecular interaction potential V(r) of dense
protein solutions under high hydrostatic pressure conditions. Small-angle X-ray scattering in combination with a
liquid-state theoretical approach was used to determine the effect of structure breaking/making salt anions
(Cl, SO4
2, PO4
3) on the intermolecular interaction of lysozyme molecules. It was found that besides the
DebyeHu¨ckel charge screening effect, reducing the repulsiveness of the interaction potential V(r) at low salt
concentrations, a specific ion effect is observed at high salt concentrations for the multivalent kosmotropic
anions, which modulates also the pressure dependence of the proteinprotein interaction potential. Whereas
sulfate and phosphate strongly influence the pressure dependence of V(r), chloride anions do not. The strong
structure-making effect of the multivalent anions, dominating for the triply charged PO4
3, renders the solution
structure less bulk-water-like at high salt concentrations, which leads to an altered behavior of the pressure
dependence of V(r). Hence, the particular structural properties of the salt solutions are able to influence the
spatial organization and the intermolecular interactions of the proteins, in particular upon compression. These
results are of interest for exploring the combined effects of ionic strength, temperature and pressure on the
phase behavior of protein solutions, but may also be of relevance for understanding pressure effects on the
hydration behavior of biological matter under extreme environmental conditions.
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Mar 2014
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[9150, 9756]
Abstract: Phase separation in ternary model membranes is known to occur over a range of temperatures and compositions and can be induced by increasing hydrostatic pressure. We have used small angle X-ray scattering (SAXS) to study phase separation along pre-determined tie lines in dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CHOL) mixtures. We can unequivocally distinguish the liquid ordered (Lo) and liquid disordered (Ld) phases in diffraction patterns from biphasic mixtures and compare their lateral compressibility. The variation of tie line endpoints with increasing hydrostatic pressure was determined, at atmospheric pressure and up to 100 MPa. We find an extension and shift of the tie lines towards the DOPC rich region of the phase diagram at increased pressure, this behaviour differs slightly from that reported for decreasing temperature.
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Nov 2015
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[14949]
Abstract: Investigating the correlation between structure and activity of oligomeric enzymes at high pressure is essential for understanding intermolecular interactions and reactivity of proteins in cellulo of organisms thriving at extreme environmental conditions as well as for biotechnological applications, such as high-pressure enzymology. In a combined experimental effort employing small-angle X-ray scattering, FT-IR and fluorescence spectroscopy as well as stopped-flow enzyme kinetics in concert with high-pressure techniques, we reveal the pressure-induced conformational changes of the dimeric enzyme horse liver alcohol dehydrogenase (LADH) on the quaternary, secondary and tertiary structural level. Moreover, the effects of cosolutes and crowding agents, mimicking intracellular conditions, have been addressed. Our results show that beyond an increase of enzymatic activity at low pressures, loss of enzyme activity occurs around 600-800 bar, i.e. in a pressure regime where small conformational changes take place in the coenzyme's binding pocket, only. Whereas higher-order oligomers dissociate at low pressures, subunit dissociation of dimeric LADH takes place, depending on the solution conditions, between 2000 and 4000 bar, only. Oligomerization and subunit dissociation are modulated by cosolvents such as urea or trimethylamine-N-oxide as well as by the crowding agent polyethylene glycol, based on their tendency to bind to the protein's interface or act via their excluded volume effect, respectively.
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Feb 2018
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[8742]
Open Access
Abstract: Can we control the crystallization of solid CaCO3 from supersaturated aqueous solutions and thus mimic a natural process predicted to occur in living organisms that produce biominerals? Here we show how we achieved this by confining the reaction between Ca2+ and CO32− ions to the environment of nanosized water cores of water-in-oil microemulsions, in which the reaction between the ions is controlled by the intermicellar exchange processes. Using a combination of in situ small-angle X-ray scattering, high-energy X-ray diffraction, and low-dose liquid-cell scanning transmission electron microscopy, we elucidate how the presence of micellar interfaces leads to the formation of a solute CaCO3 phase/species that can be stabilized for extended periods of time inside micellar water nano-droplets. The nucleation and growth of any solid CaCO3 polymorph, including the amorphous phase, from such nano-droplets is prevented despite the fact that the water cores in the used microemulsion are highly supersaturated with respect to all known calcium carbonate solid phases. On the other hand the presence of the solute CaCO3 phase inside of the water cores decreases the rigidity of the micellar surfactant/water interface, which promotes the aggregation of micelles and the formation of large (>2 μm in diameter) globules. The actual precipitation and crystallization of solid CaCO3 could be triggered “on-demand” through the targeted removal of the organic–inorganic interface and hence the destabilization of globules carrying the CaCO3 solute.
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May 2018
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[3691, 16364]
Open Access
Abstract: The structure of dispersions of TEMPO-oxidised cellulose nanofibrils (OCNF), at various concentrations, in water and in NaCl aqueous solutions, was probed using small angle X-ray scattering (SAXS). OCNF are modelled as rod-like particles with an elliptical cross-section of 10 nm and a length greater than 100 nm. As OCNF concentration increases above 1.5 wt%, repulsive interactions between fibrils are evidenced, modelled by the interaction parameter νRPA > 0. This corresponds to gel-like behaviour, where G′ > G′′ and the storage modulus, G′, shows weak frequency dependence. Hydrogels can also be formed at OCNF concentration of 1 wt% in 0.1 M NaCl(aq). SAXS patterns shows an increase of the intensity at low angle that is modelled by attractive interactions (νRPA < 0) between OCNF, arising from the screening of the surface charge of the fibrils. Results are supported by ζ potential and cryo-TEM measurements.
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May 2018
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[8594]
Open Access
Abstract: Molecular clustering and solvent–solute interactions in isopropanol solutions of fenoxycarb have been thoroughly and systematically investigated by dynamic light scattering, small-angle X-ray scattering, and nanoparticle tracking, supported by infrared spectroscopy and molecular dynamics simulations. The existence of molecular aggregates, clusters, ranging in size up to almost a micrometre is clearly recorded at undersaturated as well as supersaturated conditions by all three analysis techniques. The results systematically reveal that the cluster size increases with solute concentration and time at stagnant conditions. For most concentrations the time scale of cluster growth is of the order of days. In undersaturated solutions the size appears to eventually reach a maximum value, higher the higher the concentration. Below a certain concentration threshold clusters are significantly smaller. Clusters are found to be smaller in solutions pre-heated at a higher temperature, which offers a possible explanation for the so-called “history of solution” effect. The cluster distribution is influenced by filtration through membranes with a pore size of 0.1 μm, offering an alternative explanation for the “foreign particle-catalysed nucleation” effect. At moderate concentrations larger clusters appear to be sheared into smaller ones, but the original size distribution is rapidly re-established. At higher concentrations, although still well below solubility, the cluster size as well as solute concentration are strongly affected, suggesting that larger clusters contain at least a core of more organized molecules not able to pass through the filter.
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May 2018
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I22-Small angle scattering & Diffraction
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Abstract: Improvement of pain management strategies after arthroscopic surgery by multimodal analgesia may include the use of long-acting amide local anesthetics. Among these anesthetics, the low molecular weight local anesthetic agent bupivacaine (BUP) is attractive for use in postoperative pain management. However, it has relatively a short duration of action and imposes higher risk of systemic toxicity at relatively large bolus doses. Bupivacaine encapsulation in lipid-based delivery systems is an attractive strategy for prolonging its local anaesthetic effect and reducing the associated undesirable systemic side effects. Here, we discuss the potential development of liquid crystalline nanocarriers by using a binary lipid mixture of citrem and soy phosphatidylcholine (SPC) at different weight ratios. The produced safe-by-design family of citrem/SPC nanoparticles are attractive for use in the development of nanocarriers owing to the previously reported hemocompatibility. BUP encapsulation efficiency (EE), depending on the lipid composition , was in the range of 65-77%. In this study, nanoparticle tracking analysis (NTA) and synchrotron small-angle X-ray scattering (SAXS) were employed to gain insight into the effect of BUP solubilization and lipid composition on the size and structural characteristics of the produced citrem/SPC nanodispersions. BUP loading led to a slight change in the mean sizes (diameters) and size distributions of citrem/SPC nanoparticles. However, we found that BUP accommodation into the nanoparticles’ self-assembled interiors, triggers in BUP concentration- and lipid composition-dependent manners significant structural alterations that involve vesicles-cubosomes and vesicles-hexosomes transitions. The structural tunability of citrem/SPC nanoparticles and the implications for a potential use for intra-articular BUP delivery are discussed.
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Jun 2019
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