Macromolecular interactions in solution - Interferometric and turbidimetric studies

In the present doctoral thesis, the investigation of macromolecule interactions in three different biochemical systems is reported. Exploiting the advantages of two novel and pioneering techniques, namely biolayer interferometry and latex nanoparticle-enhanced turbidimetry, this research project uncovers a better understanding on the protein-protein interactions under study. In this dissertation, the two experimental methodologies are applied in order to characterize the binding between proteins in terms of kinetic parameters. The techniques are here described from a biochemical and analytical point of view and purification protocols of the proteins utilized in the experiments carried out are reported in detail in the Materials and methods chapter. The results of the experimental work are reported in the Results and discussion section of the dissertation. First, an investigation of the inhibiting role of lactoferrin protein in the complex formation between ACE2 receptor and SARS-CoV-2 receptor binding domain is reported. Based on computational predictions executed by applying the 2D Zernike polynomial expansion method, the binding properties of lactoferrin are identified experimentally. The same computational design was further applied to identify and manipulate a α-helix peptide that belongs to the interacting surface on ACE2 with RBD; five mutated sequences with different binding affinity were generated, and the KD experimentally observed confirmed these predictions. Finally, the multivalency feature of the ferritin protein is exercised; in the case of the ACE2-derived peptide, an engineered ferritin with one of the high-affinity peptides is expressed and purified and its binding capability with ACE2 is tested. In the final experiment described in the dissertation, the ferritin protein is modified with a PCSK9-binding molecule, in order to investigate on the hindering properties of the construct in the PCSK9 metabolic pathway. Through extensive experimentation and data analysis, this study has generated significant and promising results. Biolayer interferometry has proven to be an effective tool for quantifying protein-protein interactions with precision and real-time monitoring capabilities, revealing the kinetics and thermodynamics of these interactions in detail. Simultaneously, the use of nanoparticle-enhanced turbidimetry has expanded the understanding of macromolecular interactions. This novel approach takes advantage of the aggregation behavior of nanoparticles to enhance sensitivity and detection limits, enabling the study of molecular binding in solution in a non-invasive approach. The results obtained are very satisfactory and they set the stage for further investigations, offering the potential for new discoveries and advancements in the study of molecular interactions.