Conformational characterization of functional proteins in physiological environment by Collective Enhancement IR Absorption (CEIRA) microscopy

The functionality of a protein is interwoven with its structure, small variations in the latter may result in the arise of resistances to drugs or severe pathologies. This is one of the main reasons why the study of protein structure, field of science called structural biology, is of paramount importance and requires a multidisciplinary/multi-technique approach to understand protein structure, function and dynamics. All these analytical tools have their pros and cons, and notably no one technique alone can provide all the answers to the question of structural biology. In this field, a pivotal role is played by those techniques that elucidate the secondary structure of proteins, such as Fourier Transform Infrared (FTIR) spectroscopy. However, despite its several advantages, FTIR dramatically suffers for sensitivity limitations. The aim of my PhD is to overcome this drawback by exploiting Collective Enhanced Infrared Absorption (CEIRA) and apply it to the investigation of secondary structure of proteins of biological and biomedical relevance in aqueous environment and at ultralow concentrations. At first the main efforts were devoted to the optimization of fabrication protocols for the of CEIRA plasmonic devices. These chips are made by ordered arrays of gold nanoantennas, whose dimensions can be tuned in order to give signal enhancement in different Regions of Interest (ROIs) in the Mid-IR. Then, by using model proteins, we proceeded with the implementation of efficient strategies for the measurements of protein solutions in buffer environment and at nanomolar concentrations, in both static and dynamic conditions. Once everything was established, our objective was to study the structural modifications that the Epithelial Growth Factor Receptor, EGFR, and in particular its Kinase Domain, undergoes when in contact to a specific ligand: the tyrosine-kinase inhibitor (TKI), Lapatinib, validating the great potentialities of our technique for protein conformational analyses. In conclusion, we developed a non-destructive label-free technology for in vitro detection of structural information of protein monolayers in their native states giving the possibility to follow the dynamics of the protein binding to the surface. Our CEIRA devices are a versatile tool for further understanding of the fundamental principles underlying the mechanism on the base of protein-ligand interaction. Moreover, we believe that a platform with this kind of sensibility will open new possibilities in the study of proteins of biomedical interest, in particular for those proteins involved in diseases where missense mutations induce local conformational changes and possible misfolding, giving rise to new possibilities in the understanding of disease-related conformational changes in proteins.