Site-specific incorporation of 3-nitrotyrosine in proteins generated via genetic code expansion in E.coli

Tyrosine nitration is an oxidative post-translational protein modification relevant to various pathophysiological processes. It is potentially also involved in the redox regulation of normal cellular functions. The scientific literature provides little information on residue specificity and efficiency of nitration. In initial experiments of this thesis, we further showed that the commonly used chemical tyrosine nitration methods also lead to unwanted modifications at other amino acid residues. The multiple side reactions make it arduous to establish direct and quantitative relationships between extents of tyrosine nitration on specific sites in a protein and biological responses. Therefore, in this thesis, a novel strategy was explored, allowing incorporation of 3-nitrotyrosine (3NT) during ribosomal protein synthesis, based on a genetic code modification. It was used to generate recombinant proteins with defined 3NT-sites, in the absence of other post-translational modifications. We studied the generation and stability of the 3NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson’s disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t1/2 = 0.4 h) reduction of 3NT to 3-aminotyrosine (3AT) was observed. When screening for potential mechanisms, we found that 3NT can be reduced enzymatically to 3AT, depending on the spatial orientation of 3NT within the protein. A genetic screen of E.coli proteins, involved in the observed 3NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites, opening up new possibilities for investigating the role of tyrosine nitration in regulating protein structure and function in physiological and pathophysiological conditions. The potential reduction of the 3NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining purified nitrated protein. The findings of this thesis open for new research questions: (i) which role does the reduction of 3NT in E.coli have as a defence mechanism under inflammatory conditions? (ii) which role does 3AT play as a potential end product of potential 3NT reduction in mammalian tissue? This novel post-translational modification could be relevant in diseases related to inflammation and oxidative stress.publishe