Stability and reversibility of thermal denaturation are greatly improved by limiting terminal flexibility of Escherichia coli dihydrofolate reductase.
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 degrees C, native condition) and high temperature (80 degrees C, unfolded condition). The thermal transition of the oxidized proteins at the concentration of 0.15 mg/ml (8.5 microM) 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 0.5 mg/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 0.5 mg/ml being eluted as the high molecular aggregated form, respectively. The apparent transition temperatures (Tm) of the oxidized forms were 5-7 degrees 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 5 M 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.
Iwakura
M
M
National Institute for Advanced Interdisciplinary Research, Ibaraki.
Honda
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003553:Cystine; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*
D004269:DNA, Bacterial; D003553:Cystine; D013762:Tetrahydrofolate Dehydrogenase