Thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart.
Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.
Uchiyama
Susumu
S
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Hasegawa
Jun
J
Tanimoto
Yuko
Y
Moriguchi
Hiroshi
H
Mizutani
Masayuki
M
Igarashi
Yasuo
Y
Sambongi
Yoshihiro
Y
Kobayashi
Yuji
Y
D001419:Bacteria / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D003574:Cytochrome c Group / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D011489:Protein Denaturation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013053:Spectrophotometry; D013816:Thermodynamics
D003574:Cytochrome c Group