Mutagenic and Thermodynamic Analyses of Residual Structure in the α Subunit of Tryptophan Synthase

Abstract

The α subunit of tryptophan synthase from Escherichia coli has been previously shown to contain residual structure at 5 M urea, conditions where the secondary structure is entirely disrupted and the tyrosine residues are exposed to solvent [Saab-Rincón, G., Froebe, C. L., & Matthews, C. R. (1993) Biochemistry32, 13981−13990]. The residual structure can be monitored by one-dimensional NMR spectroscopy studies of histidine 92 whose Cε proton is sensitive to the slow exchange between this form and the unfolded protein. The temperature dependence of the cooperative urea-induced unfolding transition between intermediate and unfolded forms demonstrates that this process involves negative values for both the enthalpy and entropy changes at 25 °C. The effects of replacements of several nonpolar side chains adjacent to histidine 92 on the slopes and midpoints of the unfolding transition curve show that these side chains participate in the residual structure. A 15-residue peptide spanning histidine 92 and the mutated residues, however, is not sufficient to define this structure. These results demonstrate that the residual structure in the α subunit is stabilized by the hydrophobic effect and may involve side chains which are distant in sequence to histidine 92.