Publications

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.

Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

We introduce an analytical physical description of micro-Spatially Offset Raman Spectroscopy (micro-SORS) experiments in turbid samples with a special emphasis on its use for studying with a special emphasis on studying the penetration depth of chemical species (agents) into a matrix. Such samples exhibit varying concentration gradients with depth, and reflect typical situations encountered in Cultural Heritage framework. The experiments aim at yielding the variations (trends) of the Raman intensity ratio of the agent and matrix marker bands, H (X), with increasing micro-SORS defocusing distance (X). The physical model shows that H (X) can be expressed as a function of the concentration profiles of the analytes. The analysis makes use of Monte Carlo simulation results of the propagation of photons in turbid media. Despite the introduced approximations, the obtained formulas provide an insightful tool that allows justifying the observed trends and facilitates the interpretation of micro-SORS data and systematic comparison among different samples. Basic comparison with previously published experimental micro-SORS data indicates good consistency. The obtained relationships could also be useful for a semi-quantitative estimate of concentration profiles in unknown samples.

The majority of basaltic magmas stall in the Earth’s crust as a result of the rheological evolution caused by crystallization during transport. However, the relationships between crystallinity, rheology and eruptibility remain uncertain because it is difficult to observe dynamic magma crystallization in real time. Here, we present in-situ 4D data for crystal growth kinetics and the textural evolution of pyroxene during crystallization of trachybasaltic magmas in high-temperature experiments under water-saturated conditions at crustal pressures. We observe dendritic growth of pyroxene on initially euhedral cores, and a surprisingly rapid increase in crystal fraction and aspect ratio at undercooling ≥30 °C. Rapid dendritic crystallization favours a rheological transition from Newtonian to non-Newtonian behaviour within minutes. We use a numerical model to quantify the impact of rapid dendritic crystallization on basaltic dike propagation, and demonstrate its dramatic effect on magma mobility and eruptibility. Our results provide insights into the processes that control whether intrusions lead to eruption or not.

Solar H2O2 produced by O2 reduction provides a green, efficient, and ecological alternative to the industrial anthraquinone process and H2/O2 direct-synthesis. We report efficient photocatalytic H2O2 production at a rate of 73.4 mM h–1 in the presence of a sacrificial donor on a structurally engineered catalyst, alkali metal-halide modulated poly(heptazine imide) (MX → PHI). The reported H2O2 production is nearly 150 and >4250 times higher than triazine structured pristine carbon nitride under UV–visible and visible light (≥400 nm) irradiation, respectively. Furthermore, the solar H2O2 production rate on MX → PHI is higher than most of the previously reported carbon nitride (triazine, tri-s-triazine), metal oxides, metal sulfides, and other metal–organic photocatalysts. A record high AQY of 96% at 365 nm and 21% at 450 nm was observed. We find that structural modulation by alkali metal-halides results in a highly photoactive MX → PHI catalyst which has a broader light absorption range, enhanced light absorption ability, tailored bandgap, and a tunable band edge position. Moreover, this material has a different polymeric structure, high O2 trapping ability, interlayer intercalation, as well as surface decoration of alkali metals. The specific C≡N groups and surface defects, generated by intercalated MX, were also considered as potential contributors to the separation of photoinduced electron–hole pairs, leading to enhanced photocatalytic activity. A synergy of all these factors contributes to a higher H2O2 production rate. Spectroscopic data help us to rationalize the exceptional photochemical performance and structural characteristics of MX → PHI.

We outline procedures to calculate small-angle scattering (SAS) intensity functions from 2-dimensional electron-microscopy (EM) images. Two types of scattering systems were considered: (a) the sample is a set of particles confined to a plane; or (b) the sample is modelled as parallel, infinitely long cylinders that extend into the image plane. In each case, an EM image is segmented into particle instances and the background, whereby coordinates and morphological parameters are computed and used to calculate the constituents of the SAS-intensity function. We compare our results with experimental SAS data, discuss limitations, both general and case specific, and outline some applications of this method which could potentially complement experimental SAS.

The ability to mass produce tailored nanoparticles, e.g. uniform sizes and surface site types, and accessibilities, has wide reaching implications for both academic and industrial research. Sol-immobilisation is proven to offer an easy to control route for the preparation of metallic nanoparticles, where a colloidal solution of nanoparticles is preformed (stabilised using polyvinyl alcohol) and anchored to a support material. However, as yet, there is limited
understanding of how systematic variations to the synthesis parameters influence the fundamental nucleation and growth steps in nanoparticle formation.

Systematic changes to the solvent of synthesis, through the addition of C1-C4 linear and branched chain alcohols, were employed in the preparation of metallic Pd colloids. It was discovered using spectroscopic techniques (UV-Vis, IR and XAFS) and TEM imaging that the greatest control of nanoparticle growth was achieved in solutions of equal MeOH and H2O parts per volume. Additionally, Pd colloids prepared in solutions of > 50 vol. % MeOH
saw a drastic decrease in their achieved metal loading. This issue was remedied by removing the acidification step during immobilisation. The resultant influence of this updated procedure on Pd nanoparticle properties was characterised using the previously listed techniques along with thermal decomposition methods (TGA, TPR, TPD). From this, it was established that the synthesis of Pd nanoparticles in MeOH caused increased layering of the
stabiliser around the nanoparticle and support surfaces, suppressing the spillover of H2. The performance of these non-acidified PVA-capped Pd nanoparticles were investigated forfurfural hydrogenation. The limiting of H2 spillover was shown to be highly effective in switching off the acid-catalysation pathway to acetals over the TiO2 support surface, making it selective for the desired hydrogenation products.

In addition, the mechanisms of colloidal Au nucleation and growth were investigated via novel XAFS experiments. A proof of concept study for in situ XAFS measurements was first approached by measuring solutions of preformed Au colloids prepared under varied synthesis parameters: synthesis temperature = 1, 25, 50 and 75 °C, and [Au] = 50, 100 and 1000 µM. The established relationship between synthesis temperature and nanoparticle size was found to be consistent in colloidal and supported Au NP systems. The influence of the [Au] on XAFS nanoparticle size, however, was only observed after immobilisation of the colloid, something which has not previously been reported. Building on the successful acquisition of colloidal XAFS, a continuous microfluidic system was designed and implemented to measure the in situ reduction of HAuCl4 ([Au] = 100 µM) to Au0 nanoparticles. Issues concerning the flow regime of the cell, metal deposition on the reactor walls, and X-ray compatibility were all considered prior to in situ measurements. XAFS data acquired during reduction showed that under mild reducing conditions nanoparticle nucleation occurs on a 102 ms time scale, and that complete reduction of the Au species
occurs over the course of 3.5 seconds. Furthermore, this novel work proves that time-resolved studies of colloidal nanoparticle nucleation and growth are feasible.

Here, we demonstrate successful construction of two Fe-Metalated Porous-Organic-Polymers having planar (Fe-Tt-POP) & non-planar (Fe-Rb-POP) geometry by ternary copolymerization stratergy for catalytic oxidative decontamination of different sulfur-based mustard gas simulants (HD). Fe-Tt-POP exhibits superior catalytic performance for oxidation of the thioanisole (TA) in comparison with Fe-Rb-POP. Interestingly, this activity difference can further be explored by In-situ operando DRIFTS & DFT computational studies. Fe-Tt-POP having favorable binding interaction with the close proximity arrangement of substrates inside the catalytic core makes it good candidate for oxidative HD decontamination.

Here, we demonstrate successful construction of two Fe-Metalated Porous-Organic-Polymers having planar (Fe-Tt-POP) & non-planar (Fe-Rb-POP) geometry by ternary copolymerization stratergy for catalytic oxidative decontamination of different sulfur-based mustard gas simulants (HD). Fe-Tt-POP exhibits superior catalytic performance for oxidation of the thioanisole (TA) in comparison with Fe-Rb-POP. Interestingly, this activity difference can further be explored by In-situ operando DRIFTS & DFT computational studies. Fe-Tt-POP having favorable binding interaction with the close proximity arrangement of substrates inside the catalytic core makes it good candidate for oxidative HD decontamination.