Caratterizzazione di tecniche di marcatura di proteine per dirigere modifiche sito-selettive

Protein labelling is an essential tool for advanced biological applications, from therapeutic development to diagnostics. However, labelling techniques requires overcoming significant challenges due to protein complexity, particularly related to the presence of multiple reactive sites that can lead to undesirable multifunctionalisation, which compromises the functionality and applicability of bioconjugates. The recently developed linchpin-directed modification (LDM) method aims to address these challenges by exploiting bifunctional molecules with high chemoselectivity towards lysine and histidine residues and spacers with tuneable length. The first, reversible reaction with lysine provides an anchoring point for subsequent alkylation of histidine, occurring at distances defined by the spacer length. Our study aimed to build a platform to direct LDM based on a priori structure evaluation of species taking part in the reaction. To set up this platform Green Fluorescent Protein (GFP) was used as a model protein. We analysed the distances between reactive groups on both LDM and protein, and residues spatial orientation to predict favourable interaction. By absorption spectroscopy, we determined the solubility of a series of LDM molecules under conditions compatible with most proteins’ native state. W also calculated the reaction rates of LDM molecules with their target amino acid free in solution and within the model protein. After confirming the labelling mechanism and the modification site by mass spectrometry experiments on entire and proteolytic digestion products, we approached the validation of the predictions by creating GFP mutants lacking residues identified as potential LDM anchors to observe any changes in labelling profile respect to wild type protein. To assess the applicability of the methodological approach to proteins different from a model protein, we conjugated LDM molecules with hemoglobin, aiming to produce a bioconjugate for therapeutic uses, by employing LDM to generate chemoselectivity on hemogbobin to finally obtain a site-selective PEGylation. Additionally, the importance of site-selective modifications was further investigated by the conjugation of bovine serum albumin with a fluorescent probe, producing a highly sensitive and homogeneously biosensor, which is valuable for analytical applications such as monitoring the hydrolysis of lipid formulations used in the pharmaceutical field.