Fluorescence and folding properties of Tyr mutant tryptophan synthase α-subunits from Escherichia coli

Abstract

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase α-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to ∼40% each by Tyr 4 and Tyr 115, and to the remaining ∼20% by Tyr 173 and Tyr 175. Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by ∼40% and ∼10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu 49, and/or energy transfer between Tyrs. Two data for Y173F α-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F α-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.