Microfluidics and chemical kinetics to analyse protein interactions, aggregation, and physicochemical properties

Proteins play a major role in living systems and present a wide spectrum of functionalities. Many different types of proteins are involved into biological processes, such as the catalysis of biochemical reactions, cellular membrane transport, immune system response and DNA replication. However, some proteins and peptides might become harmful to living organisms; for example, their abnormal aggregation causes neurodegenerative disorders including Alzheimer disease (AD). One of the causes of AD is the presence of amyloid beta peptides Aβ(1-42), Aβ(1-40), which self-assemble into insoluble fibrils and plaques, which surround neuronal cells impeding synapsis. The number of AD patients is increasing, but a cure has not been founded yet. Therefore, it is crucial to investigate the mechanisms underlying amyloid aggregation and screening for compounds able to prevent this irreversible process. Microfluidics permits characterising the physicochemical properties of proteins, investigate their aggregation and study their interactions with other molecules. Chemical kinetics allows studying the microscopic events occurring during protein self-assembly. The combination of these two techniques provides a powerful tool for the identification of compounds inhibiting the aggregation process. In this thesis by using microfluidics, chemical kinetics and other biophysical assays, I have investigated the proteins isoelectric point (pI) and the inhibition of aberrant Aβ(1-42) self-assembly process. Firstly, I describe the development of a microfluidic platform allowing for the measurement of the protein pI, in a gradient-free manner. This approach overcomes a fundamental limitation of convectional techniques that is the achievement of a stable and well-controlled pH gradient. Secondly, I investigate the inhibiting effect of llama nanobodies on Aβ(1-42) aggregation. The findings from this study show that nanobodies target monomeric species with high affinity whereas interactions with fibril surfaces are weak. Finally, I discuss the use of other compounds inhibiting specific nucleation stages. These include the chaperones clusterin and brichos, as well as soot and pure carbon nanoparticles. Importantly, the addition of both chaperones to Aβ(1-42) solutions has an additive inhibitory effect on aggregation. My findings will improve the characterization of the physicochemical properties of proteins as well as providing promising candidates for the inhibition of specific stages of amyloid beta aggregation opening the way to possible cures for AD disease.Departmental Funding (Department of Chemistry, University of Cambridge

Antibiotic-derived molecular probes for bacterial imaging

Infections caused by drug resistant bacteria poses a significant threat to global human health, with predicted annual mortality of 10 million by 2050. While much attention is focused on developing better therapies, improving diagnosis would allow for rapid initiation of optimal treatment, reducing unnecessary antibiotic use and enhancing therapeutic outcomes. There are currently no whole body imaging techniques in clinical use that are capable of specifically identifying bacterial infections. We have developed antibiotic-derived fluorescent probes that bind and illuminate either Gram-positive or Gram-negative bacteria with high specificity and selectivity over mammalian cells. Antibiotics are functionalised with an azide substituent in a position that minimises effects on antibiotic activity. These are reacted by facile 1,3-dipolar cycloaddition with alkyne-substituted imaging components such as visible or near-infrared fluorophores. The resulting adducts can be used as tools to image bacteria in vitro and in vivo. We have successfully functionalised representatives of seven major antibiotic classes. These derivatives retain antibacterial activity, and have been coupled with a range of fluorophores. Fluorescent versions of vancomycin and polymyxin B are particularly useful for specific labelling of G+ve and G-ve bacteria, respectively. Preliminary studies have now extended the visualisation component to include moieties compatible with PET imaging

Secondary nucleation and elongation occur at different sites on Alzheimer\u27s amyloid-b aggregates

The aggregates of the Ab peptide associated with Alzheimer\u27s disease are able to both grow in size aswell as generate, through secondary nucleation, new small oligomeric species, that are major cytotoxins associated with neuronal death. Despite the importance of these amyloid fibril-dependent processes, their structural and molecular underpinnings have remained challenging to elucidate. Here, we consider two molecular chaperones: The Brichos domain, which suppresses specifically secondary nucleation processes, and clusterin which our results show is capable of inhibiting, specifically, the elongation of Ab fibrils at remarkably low substoichiometric ratios. Microfluidic diffusional sizing measurements demonstrate that this inhibition originates from interactions of clusterin with fibril ends with high affinity. Kinetic experiments in the presence of both molecular chaperones reveal that their inhibitory effects are additive and noncooperative, thereby indicating that the reactive sites associated with the formation of new aggregates and the growth of existing aggregates are distinct