I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[20303]
Abstract: The global HIV/AIDS epidemic still currently affects approximately 38 million individuals globally. The protease enzyme of the human immunodeficiency virus is a major drug target in antiviral therapy, however, under the influence of reverse transcriptase and in the context of drug pressure, the rapid PR mutation rate contributes significantly to clinical failure. The set of cooperative non-active site mutations, I13V/I62V/V77I, have been associated with reduced inhibitor susceptibility and are the focus of the current study. When compared to the wild-type protease the mutant protease exhibited decreased binding affinities towards ATV and DRV by 64- and 12-fold, respectively, and decreased the overall favourable Gibbs free energy for ATV, DRV, RTV and SQV. Moreover, these mutations decreased the thermal stability of the protease when in complex with ATV and DRV by approximately 6.4 and 4.2 °C, respectively. The crystal structure of the mutant protease revealed that the location of these mutations and their effect on the hydrophobic sliding mechanism may be crucial in their role in resistance.
|
Sep 2022
|
|
|
Diamond Proposal Number(s):
[29810]
Abstract: Thermoplastic elastomers (TPEs) that are closed-loop recyclable are needed in a circular material economy, but many current materials degrade during recycling, and almost all are pervasive hydrocarbons. Here, well-controlled block polyester TPEs featuring regularly placed sodium/lithium carboxylate side chains are described. They show significantly higher tensile strengths than unfunctionalized analogues, with high elasticity and elastic recovery. The materials are prepared using controlled polymerizations, exploiting a single catalyst that switches between different polymerization cycles. ABA block polyesters of high molar mass (60–100 kg mol–1; 21 wt % A-block) are constructed using the ring-opening polymerization of ε-decalactone (derived from castor oil; B-block), followed by the alternating ring-opening copolymerization of phthalic anhydride with 4-vinyl-cyclohexene oxide (A-blocks). The polyesters undergo efficient functionalization to install regularly placed carboxylic acids onto the A blocks. Reacting the polymers with sodium or lithium hydroxide controls the extent of ionization (0–100%); ionized polymers show a higher tensile strength (20 MPa), elasticity (>2000%), and elastic recovery (>80%). In one case, sodium functionalization results in 35× higher stress at break than the carboxylic acid polymer; in all cases, changing the quantity of sodium tunes the properties. A leading sample, 2-COONa75 (Mn 100 kg mol–1, 75% sodium), shows a wide operating temperature range (−52 to 129 °C) and is recycled (×3) by hot-pressing at 200 °C, without the loss of mechanical properties. Both the efficient synthesis of ABA block polymers and precision ionization in perfectly alternating monomer sequences are concepts that can be generalized to many other monomers, functional groups, and metals. These materials are partly bioderived and have degradable ester backbone chemistries, deliver useful properties, and allow for thermal reprocessing; these features are attractive as future sustainable TPEs.
|
Mar 2022
|
|
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[14892]
Abstract: RAFT dispersion polymerization of 2,2,2-trifluoroethyl methacrylate (TFEMA) is performed in n-dodecane at 90 °C using a relatively short poly(stearyl methacrylate) (PSMA) precursor and 2-cyano-2-propyl dithiobenzoate (CPDB). The growing insoluble poly(2,2,2-trifluoroethyl methacrylate) (PTFEMA) block results in the formation of PSMA–PTFEMA diblock copolymer nano-objects via polymerization-induced self-assembly (PISA). GPC analysis indicated narrow molecular weight distributions (Mw/Mn ≤ 1.34) for all copolymers, with 19F NMR studies indicating high TFEMA conversions (≥95%) for all syntheses. A pseudo-phase diagram was constructed to enable reproducible targeting of pure spheres, worms, or vesicles by varying the target degree of polymerization of the PTFEMA block at 15–25% w/w solids. Nano-objects were characterized using dynamic light scattering, transmission electron microscopy, and small-angle X-ray scattering. Importantly, the near-identical refractive indices for PTFEMA (1.418) and n-dodecane (1.421) enable the first example of highly transparent vesicles to be prepared. The turbidity of such dispersions was examined between 20 and 90 °C. The highest transmittance (97% at 600 nm) was observed for PSMA9–PTFEMA294 vesicles (237 ± 24 nm diameter; prepared at 25% w/w solids) in n-dodecane at 20 °C. Interestingly, targeting the same diblock composition in n-hexadecane produced a vesicle dispersion with minimal turbidity at a synthesis temperature of 90 °C. This solvent enabled in situ visible absorption spectra to be recorded during the synthesis of PSMA16–PTFEMA86 spheres at 15% w/w solids, which allowed the relatively weak n→π* band at 515 nm assigned to the dithiobenzoate chain-ends to be monitored. Unfortunately, the premature loss of this RAFT chain-end occurred during the RAFT dispersion polymerization of TFEMA at 90 °C, so meaningful kinetic data could not be obtained. Furthermore, the dithiobenzoate chain-ends exhibited a λmax shift of 8 nm relative to that of the dithiobenzoate-capped PSMA9 precursor. This solvatochromatic effect suggests that the problem of thermally labile dithiobenzoate chain-ends cannot be addressed by performing the TFEMA polymerization at lower temperatures.
|
Jan 2021
|
|
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[14892]
Abstract: A series of ionic diblock copolymer nanoparticles was prepared in a typical nonpolar solvent (n-dodecane) via polymerization-induced self-assembly (PISA). A single cationic repeat unit was incorporated into the poly(stearyl methacrylate) (PSMA) stabilizer of otherwise uncharged poly(stearyl methacrylate)–poly(benzyl methacrylate) (PSMA–PBzMA) diblock copolymer nanoparticles. By using short PSMA stabilizer blocks, it was possible to obtain nanoparticles with the range of morphologies expected (spheres, worms, and vesicles). For nanoparticles where all stabilizer chains possessed an ionic group, higher-order morphologies were obtained at lower PBzMA degrees of polymerization than corresponding uncharged particles, and the particles were electrophoretic. For nanoparticles where only a fraction of the stabilizer chains contained an ionic group, higher-order morphologies were obtained at precisely the same PBzMA degrees of polymerization, and the electrophoretic response was greater than when the shell was fully ionic. These particles with a partially ionic shell are a fascinating system, providing morphologies that can be predicted from the existing knowledge of the diblock copolymer morphology yet with the highest possible electrophoretic mobility.
|
Apr 2020
|
|
B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[21035]
Abstract: We report a small-angle neutron scattering (SANS) study of semidilute aqueous solutions of sodium carboxymethyl cellulose (NaCMC), in the presence of mono- (Na+) and divalent salts (Mg2+, Ca2+, Zn2+, and Ba2+). A degree of substitution of 1.3 is selected to ensure that, in salt-free solution, the polymer is molecularly dissolved. We find that Na+ and Mg2+ salt addition yields H-type phase behavior, while Ca2+, Zn2+, and Ba2+ instead yield a mixed H/L-type phase behavior dependent on the NaCMC concentration (cp), in the decreasing order of the salt concentration required to induce turbidity (at a fixed cp). Charge screening by addition of NaCl induces the disappearance of the characteristic polyelectrolyte correlation peak and eventually yields scattering profiles with a q–1 dependence over nearly 3 decades in the wavenumber q. By fitting a descriptive model to data with excess Na+, we obtain a correlation length ξ′ = 1030 cp–0.72 Å with cp in g L–1. Addition of Mg2+, which does not interact specifically with NaCMC carboxylate groups, yields an analogous screening behavior to that of Na+, albeit at lower salt concentrations, in line with its higher ionic strength. At low salt concentration, addition of specifically interacting Ca2+, Zn2+, and Ba2+ yields a comparatively greater screening of the polyelectrolyte correlation peak, and at concentrations above the phase boundary, results in excess scattering at low-q, compatible with the formation of 20–40 nm clusters. This behavior is interpreted as due to the reduction in charge density along the chain, promoting interchain association and multichain domain formation resulting in visible turbidity. Overall, drawing analogies with NaCMC at a lower degree of substitution, where hydrophobic association takes place, our findings provide a framework to describe the solution structure and phase behavior of NaCMC in salt-free and salt solutions.
|
Feb 2020
|
|
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[19852]
Abstract: Reversible addition–fragmentation chain transfer (RAFT) solution polymerization of 3-[tris(trimethylsiloxy)silyl] propyl methacrylate (SiMA) was conducted in toluene to prepare three PSiMA precursors with mean degrees of polymerization (DP) of 12, 13, or 15. Each precursor was then chain-extended in turn via RAFT dispersion polymerization of benzyl methacrylate (BzMA) in a low-viscosity silicone oil (decamethylcyclopentasiloxane, D5). 1H NMR studies confirmed that such polymerizations were relatively fast, with more than 99% BzMA conversion being achieved within 100 min at 90 °C. Moreover, gel permeation chromatography analysis indicated that these polymerizations were well controlled, with dispersities remaining below 1.25 when targeting PBzMA DPs up to 200. A phase diagram was constructed at a constant copolymer concentration of 20% w/w. Only spherical micelles were accessible when the PSiMA15 stabilizer was utilized, as determined by transmission electron microscopy and small-angle X-ray scattering (SAXS) studies. Nevertheless, these spheres exhibited narrow size distributions and tunable z-average diameters ranging between 19 and 49 nm, as determined by dynamic light scattering. In contrast, spheres, worms, or vesicles could be prepared depending on the target PBzMA DP when utilizing the relatively short PSiMA12 precursor. Moreover, each of these nano-objects could be obtained at copolymer concentrations as low as 5% w/w. To obtain more detailed structural information, these spheres, worms and vesicles were further characterized by SAXS. PSiMA12-PBzMA55 worms formed reasonably transparent free-standing gels when prepared at copolymer concentrations as low as 5% w/w and exhibited an elastic modulus (G′) of 90 Pa at 25 °C, as judged by oscillatory rheology studies. Finally, broadening of the molecular weight distribution was observed during the long-term storage of PSiMA-PBzMA dispersions at ambient temperature. We tentatively suggest that this instability is related to hydroxyl impurities in the SiMA, which leads to cross-linking side reactions. This problem also causes incipient flocculation of the spheres and worms during the long-term storage of such dispersions at 20 °C.
|
Feb 2020
|
|
B21-High Throughput SAXS
I02-Macromolecular Crystallography
|
Open Access
Abstract: Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.
|
Sep 2019
|
|
I16-Materials and Magnetism
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[11687, 14117, 15319]
Open Access
Abstract: The influence of reaction rate on the evolving polymer structure of photo-activated dimethacrylate biomedical resins was investigated using neutron and in situ synchrotron X-ray scattering with simultaneous Fourier-transform-near-infrared spectroscopy. Previous studies have correlated the degree of reactive group conversion with mechanical properties, but the impact of polymerization rate on the resultant polymer structure is unknown. Here, we demonstrate that the medium-range structural order at the functional end groups of these materials is dependent on the reaction rate. Accelerating polymerization increases correlation lengths in the methacrylate end groups but reduces the medium-range structural order per converted vinyl bond when compared with more slowly polymerized systems. At faster rates of polymerization, the conformation of atoms at the reacting end group can become fixed into the polymer structure at the onset of autodeceleration, storing residual strain. Neutron scattering confirms that the structural differences observed are reproduced at longer length scales. This effect is not as prominent in systems polymerized at slower rates despite similar final degrees of reactive group conversion. Results suggest that current interpretations of these materials, which extrapolate mechanical properties from conversion, may be incomplete. Accelerating polymerization can introduce structural differences, which will dictate residual strain and may ultimately explain the discrepancies in the predictive modeling of the mechanical behavior of these materials using conventional techniques.
|
Jul 2019
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[16258]
Abstract: Phycocyanin (PC) is the principal pigment protein in the light-harvesting antenna of cyanobacteria. Here the biochemical characterization and the 1.51 Å crystal structure of PC from cyanobacterium Nostoc sp. WR13 (Nst-PC) is reported. The P63 crystal lattice is composed of the minimal biological entities of Nst-PC, the (αβ)3 trimeric rings. The structure has been refined to R factor 11.5% (Rfree 15.4%) using anisotropic atomic B factors. A phylogenetic study shows that the α and β chains of Nst-PC are significantly clustered in a distinct clade with Acaryochloris marina. The structure was examined to look for any significant differences between Nst-PC and PC from non-desert species. Only minor differences were found in the chromophore microenvironments. The tentative energy transfer pathways in Nst-PC were modeled based on simple structural considerations.
|
May 2019
|
|
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[19852]
Abstract: A near-monodisperse monohydroxy-terminated polydimethylsiloxane (PDMS; mean degree of polymerization = 66) was esterified using a carboxylic acid-functionalized trithiocarbonate to yield a PDMS66 precursor with a mean degree of functionality of 92 ± 5% as determined by 1H NMR spectroscopy. This PDMS66 precursor was then chain-extended in turn using eight different methacrylic monomers in a low-viscosity silicone oil (decamethylcyclopentasiloxane, D5). Depending on the monomer type, such syntheses proceeded via either RAFT dispersion polymerization or RAFT emulsion polymerization. In each case the target DP of the core-forming block was fixed at 200, and the copolymer concentration was 25% w/w. Transmission electron microscopy studies indicated that kinetically trapped spheres were obtained in almost all cases. The only exception was 2-(dimethylamino)ethyl methacrylate (DMA), which enabled access to spheres, worms, or vesicles. This striking difference is attributed to the relatively low glass transition temperature for this latter block. A phase diagram was constructed for a series of PDMS66–PDMAx nano-objects by systematically increasing the PDMA target DP from 20 to 220 and varying the copolymer concentration between 10 and 30% w/w. Higher copolymer concentrations were required to access a pure worm phase, while only spheres, vesicles, or mixed phases were accessible at lower copolymer concentrations. Gel permeation chromatography studies indicated a linear evolution of number-average molecular weight (Mn) with PDMA DP while dispersities remained below 1.40, suggesting relatively well-controlled RAFT polymerizations. Small-angle X-ray scattering (SAXS) was used to characterize selected examples of spheres, worms, and vesicles. PDMS66–PDMA100–112 worms synthesized at 25–30% w/w formed free-standing gels at 20 °C. Oscillatory rheology studies performed on a 30% w/w PDMS66–PDMA105 worm dispersion indicated a storage modulus (gel strength) of 1057 Pa and a critical gelation concentration (CGC) of approximately 12% w/w. Finally, PDMS66–PDMAx worms could also be prepared in n-dodecane, hexamethyldisiloxane, or octamethylcyclotetrasiloxane. Rotational rheometry studies indicate that such worms are efficient viscosity modifiers for these nonpolar oils.
|
Mar 2019
|
|