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Protein Engineering, Vol. 15, No. 6, 455-461, June 2002
© 2002 Oxford University Press

Thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart

Susumu Uchiyama1,2, Jun Hasegawa3, Yuko Tanimoto1, Hiroshi Moriguchi1, Masayuki Mizutani4, Yasuo Igarashi4, Yoshihiro Sambongi5,6 and Yuji Kobayashi1,6

1 Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, 3 Daiichi Pharmaceutical Co., Ltd,1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, 4 Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032 and 5 Graduate School of Biosphere Sciences, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima, Hiroshima 739-8528, Japan

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}({Delta}G)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change ({Delta}CP), resulting in higher {Delta}G values over a wide temperature range (0–100°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.


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K. Oikawa, S. Nakamura, T. Sonoyama, A. Ohshima, Y. Kobayashi, S.-i. J. Takayama, Y. Yamamoto, S. Uchiyama, J. Hasegawa, and Y. Sambongi
Five Amino Acid Residues Responsible for the High Stability of Hydrogenobacter thermophilus Cytochrome c552: RECIPROCAL MUTATION ANALYSIS
J. Biol. Chem., February 18, 2005; 280(7): 5527 - 5532.
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