title: Enhanced thermodynamic stabilities of yeast iso-1-cytochromes c with amino acid replacements at positions 52 and 102.
abstract: We have determined the structures and thermodynamic stabilities of the wild type Asn-52 and unusually thermostable mutant Ile-52 yeast iso-1-cytochromes c (Das, G., Hickey, D. R. McLendon, D., McLendon, G., and Sherman, F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 496-499). Although both structures were similar, Water-166, buried within the wild type protein, is excluded from the Ile-52 mutant, which substantially reorganizes the local hydrogen bonding. Wild type Cys-102 was replaced with alanine or serine to eliminate dimerization in vitro. The Cys-102 (wild type), Ala-102, and Ser-102 proteins were equally stable, whereas the chemically modified Cys-102-SCH3 was less stable. The order of stability observed with replacements at positions 52 and 102 was as follows: Ile-52 Ala-102 greater than Ala-52 Ala-102 greater than Asn-52 Ala-102 ("normal") greater than Gly-52 Ala-102. No significant stabilization was attributed to potential energy interactions expressed as helix-forming propensities of replacements at position 52. A high correlation between differences in free energy changes and transfer free energies suggests hydrophobic interactions are the main factor for enhancing stability in the Ile-52 mutant. Additional possible contributions to the thermostability of the Ile-52 variant are energetic effects due to packing and hydrogen bonding changes surrounding position 52.
authors.affiliation: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
authors.forename: D R
authors.initials: DR
authors.lastname: Hickey
authors.forename: A M
authors.initials: AM
authors.lastname: Berghuis
authors.forename: G
authors.initials: G
authors.lastname: Lafond
authors.forename: J A
authors.initials: JA
authors.lastname: Jaeger
authors.forename: T S
authors.initials: TS
authors.lastname: Cardillo
authors.forename: D
authors.initials: D
authors.lastname: McLendon
authors.forename: G
authors.initials: G
authors.lastname: Das
authors.forename: F
authors.initials: F
authors.lastname: Sherman
authors.forename: G D
authors.initials: GD
authors.lastname: Brayer
authors.forename: G
authors.initials: G
authors.lastname: McLendon
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013057:Spectrum Analysis; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D003574:Cytochrome c Group; D007527:Isoenzymes