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Abstract: The temperature sensitivity of vaccines and therapeutic proteins forces the distribution of life‐saving treatments to rely heavily on the temperature‐controlled (usually 2 – 8 °C) supply and distribution network known as the cold chain. Here, using avidin as a model, we demonstrate how surface engineering could significantly increase the thermal stability of therapeutic proteins. A combination of spectroscopic (Fourier Transform Infrared, circular dichroism and ultraviolet‐visible) and scattering techniques (dynamic light scattering, small‐angle and wide‐angle X‐ray scattering) were deployed to probe the activity, structure, and stability of the model protein. Temperature‐dependent synchrotron radiation circular dichroism spectroscopy was used to demonstrate a significant increase in thermal stability, with a half denaturation temperature of 139.0 °C and reversible unfolding with modified avidin returning to a 90 % folded state when heated to temperatures below 100 °C. Accelerated ageing studies revealed that modified avidin retained its secondary structure after storage at 40 °C for 56 days, equivalent to 160 days at 25 °C. Furthermore, binding studies with multiple ligands revealed that the binding site remained functional after modification. As a result, this approach has potential as a storage technology for therapeutic proteins and the elimination of the cold chain, enabling dissemination of life‐saving vaccines worldwide.

Open Access

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Abstract: The amplification of molecular chirality by metal nanoparticles (NPs) is an important and rapidly evolving field in nanomaterial research with wide applications in smart materials, catalysis, and solvent–solute interactions. Here we present the results of the synthesis of gold nanoparticles (AuNPs) functionalized both with chiral ligands based on the binaphthol motif and with nematogenic groups (ChirAuLC). The materials were characterized chemically and the ratios between chiral groups and LC groups was determined. Synchrotron radiation circular dichroism (SRCD) and synchrotron based X-ray diffraction (XRD) studies show that the AuNPs favoured by the LC state arrange themselves into ordered columns and a helical superstructure appears in the mesophase of collective NPs. A specific focus has been the investigation of the chiral induction of ChirAuLC composites in two different nematic LC hosts. For a number of selected mixtures, the helical twisting power (HTP) of these NPs in systems was calculated from systematic optical observations based on optical polarizing microscopy (OPM). The experimental data show that the HTP of the investigated ChirAuLC composite is significantly larger than that of free “small molecule” chiral groups when dispersed in the same LC host and the chiral transfer efficiency of ChirAuLC is higher than NPs functionalized only with chiral groups (ChirAuNP). This is new and can be explained by a combination of a surface chirality and the domino effect of bound mesogens interacting with the bulk.

Open Access

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Abstract: Excess light causes damage to the photosynthetic apparatus of plants and algae primarily via reactive oxygen species. Singlet oxygen can be formed by interaction of chlorophyll (Chl) triplet states, especially in the Photosystem II reaction center, with oxygen. Whether Chls in the light-harvesting antenna complexes play direct role in oxidative photodamage is less clear. In this work, light-induced photobleaching of Chls in the major trimeric light-harvesting complex II (LHCII) is investigated in different molecular environments – protein aggregates, embedded in detergent micelles or in reconstituted membranes (proteoliposomes). The effects of intense light treatment were analyzed by absorption and circular dichroism spectroscopy, steady-state and time-resolved fluorescence and EPR spectroscopy. The rate and quantum yield of photobleaching was estimated from the light-induced Chl absorption changes. Photobleaching occurred mainly in Chl a and was accompanied by strong fluorescence quenching of the remaining unbleached Chls. The rate of photobleaching increased by 140% when LHCII was embedded in lipid membranes, compared to detergent-solubilized LHCII. Removing oxygen from the medium or adding antioxidants largely suppressed the bleaching, confirming its oxidative mechanism. Singlet oxygen formation was monitored by EPR spectroscopy using spin traps and spin labels to detect singlet oxygen directly and indirectly, respectively. The quantum yield of Chl a photobleaching in membranes and detergent was found to be 3.4 × 10–5 and 1.4 × 10–5, respectively. These values compare well with the yields of ROS production estimated from spin-trap EPR spectroscopy (around 4 × 10–5 and 2 × 10–5). A kinetic model is proposed, quantifying the generation of Chl and carotenoid triplet states and singlet oxygen. The high quantum yield of photobleaching, especially in the lipid membrane, suggest that direct photodamage of the antenna occurs with rates relevant to photoinhibition in vivo. The results represent further evidence that the molecular environment of LHCII has profound impact on its functional characteristics, including, among others, the susceptibility to photodamage.

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Abstract: Lipidic bicelles have been widely used for the analysis of integral membrane proteins where their spontaneous alignment in an magnetic field has been exploited for oriented sample solid-state NMR studies. Many of their physical properties however make them well suited to the analysis of membrane proteins by circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD). In this paper we have identified bicelle compositions that permit comparable studies of integral membrane proteins by solid-state NMR, CD, and SRCD; complementary methods that can provide insights into protein structure and the interactions with drugs and other small molecules. Furthermore we have been able to identify bicelle compositions that allow the magnetic alignment of the bicelles at fields routinely available in magnetic CD instruments. This potentially provides a route to the preparation of samples for oriented CD that mitigates many of the issues associated with the preparation of mechanically aligned samples, although we demonstrate that the dynamics present within the system can complicate the analysis of such spectra.

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Abstract: Dedicated chemistries for on-demand capture and release of biomolecules at the solid–liquid interface are required for applications in drug delivery, for the synthesis of switchable surfaces used in analytical devices and for the assembly of next-generation biomaterials with complex architectures and functions. Here we report the engineering of a binary self-assembling polypeptide system for reversible protein capture, immobilisation and controlled thermo-responsive release from a solid surface. The first element of the binary system is a universal protein substrate immobilised on a solid surface. This protein is bio-inspired by the neuronal SNAP25, which is the protein involved in the docking and fusion of synaptic vesicles to the synaptic membrane. The second element is an artificial chimeric protein engineered to include distinct domains from three different proteins: Syntaxin, VAMP and SNAP25. These native proteins constitute the machinery dedicated to vesicle trafficking in eukaryotes. We removed approximately 70% of native protein sequence from these proteins and constructed a protein chimera capable of high affinity interaction and self-assembly with immobilised substrate. The interaction of the two parts of the engineered protein complex is strong but fully-reversible and therefore the chimera can be recombinantly fused as a tag to a protein of interest, to allow spontaneous assembly and stimuli-sensitive release from the surface upon heating at a predetermined temperature. Two thermo-responsive tags are reported: the first presents remarkable thermal stability with melting temperature of the order of 80 °C; the second disassembles at a substantially lower temperature of about 45 °C. The latter is a promising candidate for remote-controlled localised delivery of therapeutic proteins, as physiologically tolerable local increase of temperatures in the 40–45 °C range can be achieved using magnetic fields, infra-red light or focused ultrasound. Importantly, these two novel polypeptides provide a broader blueprint for the engineering of future functional proteins with predictable folding and response to external stimuli.