I04-1-Macromolecular Crystallography (fixed wavelength)
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Daniel J
Shaw
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Lorna C.
Waters
,
Sarah L
Strong
,
Monika-Sarah E. D.
Schulze
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Gregory M
Greetham
,
Michael
Towrie
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Anthony W.
Parker
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Christine E.
Prosser
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Alistair J.
Henry
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Alistair D. G.
Lawson
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Mark D.
Carr
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Richard J.
Taylor
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Neil T.
Hunt
,
Frederick W
Muskett
Diamond Proposal Number(s):
[29404]
Open Access
Abstract: Knowledge of protein dynamics is fundamental to the understanding of biological processes, with NMR and 2D-IR spectroscopy being two of the principal methods for studying protein dynamics. Here, we combine these two methods to gain a new understanding of the complex mechanism of a cytokine:receptor interaction. The dynamic nature of many cytokines is now being recognised as a key property in the signalling mechanism. Interleukin-17’s (IL-17) are proinflammatory cytokines which, if unregulated, are associated with serious autoimmune diseases such as psoriasis, and although there are several therapeutics on the market for these conditions, small molecule therapeutics remain elusive. Previous studies, exploiting crystallographic methods alone, have been unable to explain the dramatic differences in affinity observed between IL-17 dimers and their receptors, suggesting there are factors that cannot be fully explained by the analysis of static structures alone. Here, we show that the IL-17 family of cytokines have varying degrees of flexibility which directly correlates to their receptor affinities. Small molecule inhibitors of the cytokine:receptor interaction are usually thought to function by either causing steric clashes or structural changes. However, our results, supported by other biophysical methods, provide evidence for an alternate mechanism of inhibition, in which the small molecule rigidifies the protein, causing a reduction in receptor affinity. The results presented here indicate an induced fit model of cytokine:receptor binding, with the more flexible cytokines having a higher affinity. Our approach could be applied to other systems where the inhibition of a protein-protein interaction has proved intractable, for example due to the flat, featureless nature of the interface. Targeting allosteric sites which modulate protein dynamics, opens up new avenues for novel therapeutic development.
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Jun 2023
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I11-High Resolution Powder Diffraction
I19-Small Molecule Single Crystal Diffraction
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Guo-Hong
Ning
,
Peng
Cui
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Igor V.
Sazanovich
,
James T.
Pegg
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Qiang
Zhu
,
Zhongfu
Pang
,
Rong-Jia
Wei
,
Mike
Towrie
,
Kim E.
Jelfs
,
Marc A.
Little
,
Andrew I.
Cooper
Diamond Proposal Number(s):
[21726, 17193]
Abstract: Host-guest complexation is an important supramolecular route to materials. Clear design rules have been developed for complexation in solution. This has proved more challenging for solid-state host-guest co-crystals because they often exhibit polymorphism, leading many researchers to focus instead on bonded frameworks, such as metal-organic frameworks. Here, we report an anthracene-based organic cage (1) that forms isoskeletal host-guest co-crystals with five similarly sized solid organic guests. The co-crystals were designed using inexpensive computational methods to identify appropriate guests that have packing coefficients (PCs) ranging from 44% to 50%, coupled with consideration of the guest shape. By complexing highly emissive BODIPY guests into the host structure, we enhanced its two-photon excited photoluminescent properties by a factor of six. Our crystal design approach was also transferrable to hard-to-design ternary organic crystals that were accessed by inserting specific guests into different sized voids in the host.
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Oct 2021
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I04-Macromolecular Crystallography
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Jessica H.
Van Wonderen
,
Katrin
Adamczyk
,
Xiaojing
Wu
,
Xiuyun
Jiang
,
Samuel E. H.
Piper
,
Christopher R.
Hall
,
Marcus J.
Edwards
,
Thomas A.
Clarke
,
Huijie
Zhang
,
Lars J. C.
Jeuken
,
Igor V.
Sazanovich
,
Michael
Towrie
,
Jochen
Blumberger
,
Stephen R.
Meech
,
Julea N.
Butt
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 μm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme–heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis. We observed rates of heme-to-heme electron transfer on the order of 109 s−1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser–Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-μs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.
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Sep 2021
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Abstract: Ultrafast time resolved infrared (TRIR) is used to report on the binding site of the “light-switch” complex [Ru(phen)2(dppz)]2+ 1 to i-motif structures in solution. Detailed information is provided due to perturbation of the local base vibrations by a ‘Stark-like’ effect which is used to establish the contribution of thymine base loop interactions to the binding site of 1 in this increasingly relevant DNA structure.
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Jul 2020
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Stuart A.
Bartlett
,
Nicholas A.
Besley
,
Andrew J.
Dent
,
Sofia
Diaz-Moreno
,
John
Evans
,
Michelle L.
Hamilton
,
Magnus W. D.
Hanson-Heine
,
Raphael
Horvath
,
Valentina
Manici
,
Xue-Zhong
Sun
,
Michael
Towrie
,
Lingjun
Wu
,
Xiaoyi
Zhang
,
Michael W.
George
Abstract: Complexes with weakly coordinating ligands are often formed in chemical reactions and can play key roles in determining the reactivity, particularly in catalytic reactions. Using time-resolved X-ray absorption fine structure (XAFS) spectroscopy in combination with time-resolved IR (TRIR) spectroscopy and tungsten hexacarbonyl, W(CO)6, we are able to structurally characterize the formation of an organometallic alkane complex, determine the W–C distances, and monitor the reactivity with silane to form an organometallic silane complex. Experiments in perfluorosolvents doped with xenon afford initially the corresponding solvated complex, which is sufficiently reactive in the presence of Xe that we can then observe the coordination of Xe to the metal center, providing a unique insight into the metal–xenon bonding. These results offer a step toward elucidating the structure, bonding, and chemical reactivity of transient species by X-ray absorption spectroscopy, which has sensitivity to small structural changes. The XAFS results indicate that the bond lengths of metal–alkane (W–H–C) bond in W(CO)5(heptane) as 3.07 (±0.06) Å, which is longer than the calculated W–C (2.86 Å) for binding of the primary C–H, but shorter than the calculated W–C (3.12 Å) for the secondary C–H. A statistical average of the calculated W–C alkane bond lengths is 3.02 Å, and comparison of this value indicates that the value derived from the XAFS measurements is averaged over coordination of all C–H bonds consistent with alkane chain walking. Photolysis of W(CO)6 in the presence of HSiBu3 allows the conversion of W(CO)5(heptane) to W(CO)5(HSiBu3) with an estimated W–Si distance of 3.20 (±0.03) Å. Time-resolved TRIR and XAFS experiments following photolysis of W(CO)6 in perfluoromethylcyclohexane (PFMCH) allows the characterization of W(CO)5(PFMCH) with a W–F distance of 2.65 (±0.06) Å, and doping PFMCH with Xe allows the characterization of W(CO)5Xe with a W–Xe bond length of 3.10 (±0.02) Å.
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Jul 2019
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[13641, 16214]
Abstract: Understanding defect formation during laser additive manufacturing (LAM) of virgin, stored, and reused powders is crucial for the production of high quality additively manufactured parts. We investigate the effects of powder oxidation on the molten pool dynamics and defect formation during LAM. We compare virgin and oxidised Invar 36 powder under overhang and layer-by-layer build conditions using in situ and operando X-ray Imaging. The oxygen content of the oxidised powder was found to be ca. 6 times greater (0.343 wt.%) than the virgin powder (0.057 wt.%). During LAM, the powder oxide is entrained into the molten pool, altering the Marangoni convection from an inward centrifugal to an outward centripetal flow. We hypothesise that the oxide promotes pore nucleation, stabilisation, and growth. We observe that spatter occurs more frequently under overhang conditions compared to layer-by-layer conditions. Droplet spatter can be formed by indirect laser-driven gas expansion and by the laser-induced metal vapour at the melt surface. In layer-by-layer build conditions, laser re-melting reduces the pore size distribution and number density either by promoting gas release from keyholing or by inducing liquid flow, partially or completely filling pre-existing pores. We also observe that pores residing at the track surface can burst during laser re-melting, resulting in either formation of droplet spatter and an open pore or healing of the pore via Marangoni flow. This study confirms that excessive oxygen in the powder feedstock may cause defect formation in LAM.
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Dec 2018
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I12-JEEP: Joint Engineering, Environmental and Processing
I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[13641, 15250]
Open Access
Abstract: Laser-matter interactions in laser additive manufacturing (LAM) occur on short time scales (10-6 - 10-3 s) and have traditionally proven difficult to characterise. We investigate these interactions during LAM of stainless steel (SS316 L) and 13-93 bioactive glass powders using a custom built LAM process replicator (LAMPR) with in situ and operando synchrotron X-ray radiography. This reveals a range of melt track solidification phenomena as well as spatter and porosity formation. We hypothesise that the SS316 L powder absorbs the laser energy at its surface while the trace elements in the 13-93 bioactive glass powder absorb the laser energy by radiation conduction. Our results show that a low viscosity melt, e.g. 8 mPa s for SS316 L, tends to generate spatter with a diameter up to 250 µm and an average spatter velocity of 0.26 m s-1 and form a melt track by molten pool wetting. In contrast, a high viscosity melt, e.g. 2 Pa s for 13-93 bioactive glass, inhibits spatter formation by damping the Marangoni convection, forming a melt track via viscous flow. The viscous flow in 13-93 bioactive glass resists pore transport; combined with the reboil effect, this promotes pore growth during LAM, resulting in a pore size up to 500 times larger than that exhibited in the SS316 L sample.
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Aug 2018
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[11761]
Open Access
Abstract: The laser–matter interaction and solidification phenomena associated with laser additive manufacturing (LAM) remain unclear, slowing its process development and optimisation. Here, through in situ and operando high-speed synchrotron X-ray imaging, we reveal the underlying physical phenomena during the deposition of the first and second layer melt tracks. We show that the laser-induced gas/vapour jet promotes the formation of melt tracks and denuded zones via spattering (at a velocity of 1 m s−1). We also uncover mechanisms of pore migration by Marangoni-driven flow (recirculating at a velocity of 0.4 m s−1), pore dissolution and dispersion by laser re-melting. We develop a mechanism map for predicting the evolution of melt features, changes in melt track morphology from a continuous hemi-cylindrical track to disconnected beads with decreasing linear energy density and improved molten pool wetting with increasing laser power. Our results clarify aspects of the physics behind LAM, which are critical for its development.
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Apr 2018
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B23-Circular Dichroism
I02-Macromolecular Crystallography
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John M.
Kelly
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Paraic
Keane
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James P.
Hall
,
Fergus
Poynton
,
Bjorn
Poulsen
,
Sarah P.
Gurung
,
Ian P.
Clark
,
Igor
Sazanovich
,
Michael
Towrie
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Thorfinnur
Gunnlaugsson
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Susan J.
Quinn
,
Christine J.
Cardin
Diamond Proposal Number(s):
[9684, 11291]
Abstract: Key to the development of DNA-targeting phototherapeutic drugs is determining the interplay between the photoactivity of the drug and its binding preference for a target sequence. For the photo-oxidising lambda-[Ru(TAP)2(dppz)]2+ (Ʌ-1) complex bound to either d{T1C2G3G4C5G6C7C8G9A10}2 (G9) or d{TCGGCGCCIA}2 (I9), the X-ray crystal structures shows the dppz intercalated at the terminal T1C2;G9A10 step or T1C2;I9A10 step. Thus substitution of the G9 nucleobase by inosine does not affect intercalation in the solid state although with I9 the dppz is more deeply inserted. In solution it is found that the extent of guanine photo-oxidation, and the rate of back electron transfer, as determined by ps and ns time-resolved infrared and transient visible absorption spectroscopy, is enhanced in I9, despite it containing the less oxidisable inosine. This is attributed to the nature of the binding in the minor groove due to the absence of an NH2 group. Similar behaviour and the same binding site in the crystal.are found for d{TTGGCGCCAA}2 (A9), In solution we propose that intercalation occurs at the C2G3;C8I9 or T2G3;C8A9 steps, respectively, with G3 the likely target for photo-oxidation. This demonstrates how changes in the minor groove (in this case removal of an NH2 group) can facilitate binding of Ru(II)dppz complexes and hence influence any sensitised reactions occurring at these sites. No similar enhancement of photooxidation on binding to I9 is found for the delta enantiomer.
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May 2017
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Fergus
Poynton
,
James P.
Hall
,
Paraic
Keane
,
Christine
Schwarz
,
Igor V.
Sazanovich
,
Mike
Towrie
,
Thorfinnur
Gunnlaugsson
,
Christine J
Cardin
,
David
Cardin
,
Susan J.
Quinn
,
Conor
Long
,
John
Kelly
Open Access
Abstract: The [Ru(phen)2(dppz)]2+ complex (1) is non-emissive in water but is highly luminescent in organic solvents or when bound to DNA, making it a useful probe for DNA binding. To date, a complete mechanistic explanation for this “light-switch” effect is still lacking. With this in mind we have undertaken an ultrafast time resolved infrared (TRIR) study of 1 and directly observe marker bands between 1280–1450 cm−1, which characterise both the emissive “bright” and the non-emissive “dark” excited states of the complex, in CD3CN and D2O respectively. These characteristic spectral features are present in the [Ru(dppz)3]2+ solvent light-switch complex but absent in [Ru(phen)3]2+, which is luminescent in both solvents. DFT calculations show that the vibrational modes responsible for these characteristic bands are predominantly localised on the dppz ligand. Moreover, they reveal that certain vibrational modes of the “dark” excited state couple with vibrational modes of two coordinating water molecules, and through these to the bulk solvent, thus providing a new insight into the mechanism of the light-switch effect. We also demonstrate that the marker bands for the “bright” state are observed for both Λ- and Δ-enantiomers of 1 when bound to DNA and that photo-excitation of the complex induces perturbation of the guanine and cytosine carbonyl bands. This perturbation is shown to be stronger for the Λ-enantiomer, demonstrating the different binding site properties of the two enantiomers and the ability of this technique to determine the identity and nature of the binding site of such intercalators.
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Jan 2016
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