The incorporation of amino acid analogs bearing new chemical functionalities expands the protein engineering capabilities with tremendous potential for biotechnology including therapeutic design. Among the variety of unnatural amino acids incorporated into proteins, fluorinated residues demonstrate distinct properties dictated by the presence of the highly electronegative and hydrophobic fluorine atom. Greater thermal stability and resistance to chemical denaturation has been demonstrated for proteins containing trifluorovaline, trifluoroleucine, trifluoroisoleucine, fluoroproline and hexafluoroleucine. However, the presence of fluorinated amino acids in certain large globular proteins, have led to reduction in stability or function. Thus, there is a context dependence of the influence of fluorinated analogs on proteins. Although the effects of globally incorporated saturated fluorocarbon analogs have been largely studied, there are few examples investigating the influence of fluorinated aromatic side chains on protein structure and function. To investigate the positional effects of fluorine substitution on aromatic side chains in biologically relevant globular proteins, we chose to explore the effects of monofluorinated phenylalanine analogs on histone acetyltransferases (HATs). Here we study the influence of residue-specific incorporation of para-fluorophenylalanine (pFF), meta-fluorophenylalanine (mFF), and ortho-fluorophenylalanine (oFF) on the structure and fucntion of the HAT protein, tGCN5. Although the tGCN5 bearing monofluorinated analogs resulted in as much as 10 fold loss in activity relative to wildtype and loss in stability, the fluorinated proteins exhibited an enhanced selectivity for histone substrate over the non-histone, p53. These studies demonstrate that although oFF, oFF and mFF are isomers leading to an equal extent of protein fluorination, the position of the fluorine group plays a critical role in its folding, stability and function.

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