Enhanced thermodynamic stabilities of yeast iso-1-cytochromes c with amino acid replacements at positions 52 and 102.
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.
Hickey
D R
DR
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Berghuis
A M
AM
Lafond
G
G
Jaeger
J A
JA
Cardillo
T S
TS
McLendon
D
D
Das
G
G
Sherman
F
F
Brayer
G D
GD
McLendon
G
G
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
D000596:Amino Acids; D003574:Cytochrome c Group; D007527:Isoenzymes