New Reagents for Protein Modification

Cysteine oxidation has attracted increasing interest recently, due to cumulative evidence of its key role in several critical and complex cellular processes, notably oxidative stress response mechanisms and redox cellular signalling pathways. It is estimated that up to 12% of all cysteines in the human proteome is in an oxidised form in cells and tissues. Astonishingly, cysteine sulfinic acid (Cys-SO2H, CSA) alone is thought to account for ~5% of accessible (i.e. not buried in the protein core) cysteines in the human proteome. While increasing evidence in recent years have suggested that CSA acts as a regulatory modification, the full scope of its biological formation and activity remains poorly understood. A major obstacle is the lack of methods to incorporate CSA chemically within purified proteins to enable accurate biochemical studies. Indeed, current methods for the preparation of CSA have proven extremely laborious and have relied on treating a cysteine thiol (Cys-SH) containing peptide/protein of interest (POI) with oxidising agents such as hydrogen peroxide or peroxyacids. Such methods are particularly inefficient and produce complex mixtures of oxidised POIs, further hampering purification and accurate characterisation in biochemical and biophysical assays. To address this critical caveat, we aim to develop new methods allowing the incorporation of CSA within recombinant proteins for functional studies, by modulating the nucleophilicity hence reactivity of the cysteine thiol towards oxidation. This programme of work will involve two main aspects: First, it will establish the design and synthetic routes towards novel cysteine chemoselective reagents in a CAPPING-OXIDATION-UNCAPPING one-pot sequence, along with biocompatible protocols for the efficient conversion of cysteine to CSA in short peptides. Second, we will then take advantage of these new reagents/protocols for the controlled introduction of CSA in an array of recombinant, therapeutically relevant proteins in proof-of-concept experiments. An important focus of our investigation will be directed at shedding initial light on the influence of CSA on the structure, stability and molecular recognition properties of proteins. Overall, the successful development of these methodologies will provide the community with a valuable new tools to investigate the function and relevance of CSA in human physiology and disease. Finally, it will also open exciting avenues for the development of new generations of synthetic proteins/peptides encoding new activities and functions, for both fundamental science and therapeutic applications.