Protein Engineering, CRISPR-based diagnostics, CRISPR Cas enzymes

Thesis: “Biochemical and structural study of the assembly CuA site from Arabidopsis thaliana”Supervisor: Prof. Alejandro J. VilaDuring my PhD, I worked in the mechanisms of biogenesis of Cytochrome c Oxidase from Arabidopsis thaliana, particularly, the assembly of a copper site (CuA). With this aim I first designed and engineered a chimeric protein (Tt3LAt), which mimics the CoxII subunit of A. thaliana, being stable in solution. I achieve this by engineering the functional part of A. thaliana CoxII into the thermostable scaffold from Thermus thermophilus’ homolog protein. I characterized spectroscopically and functionally this protein in its apo and copper-bound forms. I performed and analyzed the electronic absorption and EPR spectra of the oxidized CuA protein and characterized the site. I also assigned and analyzed the paramagnetically shifted NMR signals from the coordinating residues. Working in collaboration we also solved the copper-bound crystal structure of the proteins (PDB 6PTT). The analysis of all this data revealed novel features about the electron transfer pathways in CuA proteins (Morgada et al., ChemComm, 2020). Afterward, I expressed and purified a putative assembly factor of A. thaliana Cox II (Hcc1). I showed that It is a copper-binding protein, being able to bind both Cu(I) and Cu(II) with high affinity. I characterized spectroscopically (by UV-Vis, CD, NMR, EPR) the Cu(II) binding site and, in collaboration, we solved the structure of the Cu(I)-bound form (PDB 6N5U). Finally, I assessed the functional features of this protein by in vitro NMR experiments. I showed that it is a copper metallochaperone, inserting two copper ions into the CuA site from Tt3LAt (Llases et al., FEBS J. 2019).

Protein team - CRISPR-based COVID-19 diagnostics

Research visit to Merce Capdevila's Laboratory to perform Mass Spectrometry experiments.

In my Undergraduate Thesis, and afterwards, in a research period in the same laboratory, I worked in structural biology of amyloid proteins, specifically Alpha-Synuclein (AS). AS is an amyloid-forming intrinsically disordered protein (IDP) that has been associated with the etiology of Parkinson Disease. I used biomolecular NMR in order to assess the binding mode and mechanism of action of anti-amyloid potential drugs, particularly a series of apo and metal-bound porphyrins. I mapped the interaction sites of these compounds in the protein. I also calculated the relative affinities of such sites, contributing to the development of a hypothesis on the mechanism of amyloid inhibition by these compounds. In a second project in the same laboratory, I designed a series of AS variants with the aim of identifying the relevance of the aromaticity of in the process of amyloid formation. Pi-stacking interactions among aromatic residues had been reported as key first contacts in the onset of amyloid formation. Particularly, I wanted to analyse the relative contribution of aromatic and hydrophobic interactions in the mechanism of aggregation of AS. I measured aggregation kinetics of these variants and also studied their structural features by biomolecular NMR. I identified differential inter- and intra-molecular interactions for variants of AS by using a Paramagnetic Relaxation Enhancement (PRE) NMR technique. I attached covalently a paramagnetic tag (MTSL) in different sequence positions and calculated the distances from the tag to different regions of the protein based on the changes in relaxation rated of the NMR signals. These analyses revealed novel details about the mechanism of aggregations of AS, and opened new perspectives for the rational design of inhibitors.