caa a22 4500 39755818X CHVBK 20180308164809.0 cr unu---uuuuu 161202e199603 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021257 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021257 Stability and Reversibility of Thermal Denaturation Are Greatly Improved by Limiting Terminal Flexibility of Escherichia coli Dihydrofolate Reductase [Elektronische Daten] [Masahiro Iwakura, Shinya Honda] 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 (8.5 μM) 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° 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. © by the Journal of Biochemistry Regular Papers nationallicence circularized protein nationallicence dihydrofolate reductase nationallicence Escherichia coli nationallicence reversibility nationallicence thermal stability nationallicence Iwakura Masahiro National Institute for Advanced Interdisciplinary Research 1-1-4 Higashi, Tsukuba, Ibaraki 305 aut Honda Shinya National Institute for Advanced Interdisciplinary Research 1-1-4 Higashi, Tsukuba, Ibaraki 305 aut The Journal of Biochemistry Oxford University Press 119/3(1996-03), 414-420 0021-924X 119:3<414 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021257 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021257 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Iwakura Masahiro National Institute for Advanced Interdisciplinary Research 1-1-4 Higashi, Tsukuba, Ibaraki 305 aut NATIONALLICENCE 700 1- Honda Shinya National Institute for Advanced Interdisciplinary Research 1-1-4 Higashi, Tsukuba, Ibaraki 305 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/3(1996-03), 414-420 0021-924X 119:3<414 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford