Enhancing the fluorescence of tyr-59 in ubiquitin by blocking proton transfer

Abstract

Two highly fluorescent mutants of ubiquitin (E51Q and E51A) were produced by mutation of a single amino acid, demonstrating that excited-state proton transfer from the tyrosine residue to the carboxylate group of Glu-51 in ubiquitin is responsible for the reduced fluorescence of wild-type ubiquitin (wt-UBQ) at pH 5. E51A shows a T_m = 59 °C at pH 1.5 and a T_m > 80 °C at pH 5 similar to wt-UBQ, which shows that the mutation has not altered the protein structure significantly. The high and constant fluorescence from pH 1.5 to pH 7 allows for the study of the folding/unfolding over a wide range of pHs which would otherwise be impossible with wt-UBQ.