Stability and Reversibility of Thermal Denaturation Are Greatly Improved by Limiting Terminal Flexibility of Escherichia coli Dihydrofolate Reductase

抄録

Short peptides which contained a single Cys residue were introduced into both N- and C-termini of the Cys-free mutant of DHFR (Cys85→Ala, Cys152→Ser double mutant) by a recombinant DNA method, then the terminal regions were connected through a disulfide bond by oxidation. The oxidized form and reduced form proteins have as high enzymatic activity as wild-type DHFR. There is no detectable difference between the CD spectra of the reduced and oxidized forms at low (15°C, native condition) and high temperature (80°C, unfolded condition). The thermal transition of the oxidized proteins at the concentration of 0.15mg/ml (8.5μM) is completely reversible as demonstrated by the CD spectra. No aggregated materials were detected in the oxidized protein on gel-filtration HPLC after heat treatment up to the protein concentration of 0.5mg/ml. The reduced protein, however, even in the presence of reducing agent, showed only partial reversibility, with as much as 55 and 95% of the heat-treated protein at the concentrations of 0.15 and 0.5mg/ml being eluted as the high molecular aggregated form, respectively. The apparent transition temperatures (T_m) of the oxidized forms were 5-7°C higher than those of the reduced counterparts. The oxidized protein that had been denatured with guanidine-HCl was eluted later than the denatured reduced protein on gel-filtration HPLC in the presence of 5M guanidine-HCl. The limitation of spatial movement of the termini may prevent intermolecular interaction of exposed domains during denaturation-renaturation process, giving rise to the irreversible denaturation. The flexibility of the terminal is also suggested to be an important factor for improving thermal stability of proteins.