Careful scrutiny of the protein interior of @entity1361 cytochrome c(552) ( @entity101 ) on the basis of its X-ray structure [Travaglini-Allocatelli, C., Gianni, S., Dubey, V. K., Borgia, A., Di Matteo, A., Bonivento, D., Cutruzzola, F., Bren, K. L., and Brunori, M. (2005) J. Biol. Chem. 280, 25729-25734] indicated that a void space, which is large enough to accommodate a @entity5498 group, exists in the hydrophobic protein interior near the @entity1446 . We tried to reduce the void space through the replacement of a @entity2596 by Ile or Leu ( @entity2596 /Ile or @entity2596 /Leu mutation), and then the structural and functional consequences of these two mutations were characterized in order to elucidate the relationship between the nature of the packing of hydrophobic residues and the functional properties of the protein. The study demonstrated striking differences in the structural and functional consequences between the two mutations. The @entity2596 /Ile mutation was found to cause further enhancement of the thermostability of the oxidized @entity101 , as reflected in the increase of the denaturation temperature (T(m)) value by     3 deg, whereas the thermostability of the reduced form was essentially unaffected. As a result, the redox potential (E(m)) of the @entity2596 /Ile mutant exhibited a negative shift of     50 mV relative to that of the wild-type protein in an enthalpic manner, this being consistent with our previous finding that a protein with higher stability in its oxidized form exhibits a lower E(m) value [Terui, N., Tachiiri, N., Matsuo, H., Hasegawa, J., Uchiyama, S., Kobayashi, Y., Igarashi, Y., Sambongi, Y., and Yamamoto, Y. (2003) J. Am. Chem. Soc. 125, 13650-13651]. In contrast, the @entity2596 /Leu mutation led to a decrease in thermostability of both the redox forms of the protein, as reflected in the decreases of the T(m) values of the oxidized and reduced proteins by     3 and     5 deg, respectively, and the E(m) value of the @entity2596 /Leu mutant happened to be similar to that of the @entity2596 /Ile one. The E(m) value of the @entity2596 /Leu mutant could be reasonably interpreted in terms of the different effects of the mutation on the stabilities of the two different redox forms of the protein. Thus, the present study demonstrated that the stability of the protein is affected quite sensitively by the contextual stereochemical packing of hydrophobic residues in the protein interior and that the structural properties of the hydrophobic core in the protein interior are crucial for control of the redox function of the protein. These findings provide novel insights as to functional control of a protein, which could be utilized for tuning of the T(m) and E(m) values of the protein by means of protein engineering.

Enhancement of the thermostability of XXXX cytochrome c(552) through introduction of an extra methylene group into its hydrophobic protein interior.

@entity101 :: ('MESH:D006973', 'Disease') :: ['HT'] @entity2596 :: ('CHEBI:16414', 'Chemical') :: ['Val'] @entity5498 :: ('CHEBI:32875', 'Chemical') :: ['methyl'] @entity1361 :: ('940', 'Species') :: ['Hydrogenobacter thermophilus'] @entity1446 :: ('MESH:D006418', 'Chemical') :: ['heme']
@entity1361 :: (940,Species) :: ['Hydrogenobacter thermophilus']
