Construction and characterization of mutant iso-2-cytochromes c with replacement of conserved prolines.
Oligonucleotide-directed mutagenesis has been used to construct two mutant forms of iso-2-cytochrome c. In one, Pro-30 is replaced by threonine; in the other, Pro-76 is replaced by glycine. Both prolines are fully conserved among mitochondrial cytochromes c and play important structural and functional roles. Yeast with either the Pro-30 or the Gly-76 mutation has appreciable levels of mutant protein in vivo and grows on media containing nonfermentable carbon sources. Thus, neither mutation blocks protein targeting to mitochondria, uptake by mitochondria, covalent attachment of heme, or in vivo function. As judged by ultraviolet-visible spectrophotometry and proton nuclear magnetic resonance spectroscopy, the nativelike conformation of purified Gly-76 iso-2 at pH 6 is almost indistinguishable from that of the normal protein at pH 6. Ultraviolet second-derivative spectrophotometry, however, suggests an increase in the average number of exposed tyrosine side chains, with 2.25 out of 5 residues exposed for the mutant compared to 1.95 for normal iso-2. Above neutral pH, the protein folds to a mutant conformation possibly related to alkaline cytochrome c. Nuclear Overhauser difference spectroscopy of the reduced nativelike conformation allows assignment of several proton resonances and comparison of side-chain conformations of the heme ligand Met-80 in the mutant and the normal proteins. The proton chemical shifts for the assigned resonances are the same within errors for Gly-76 iso-2 and normal iso-2 at pD 6, 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Wood
L C
LC
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Muthukrishnan
K
K
White
T B
TB
Ramdas
L
L
Nall
B T
BT
D000595:Amino Acid Sequence; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D045304:Cytochromes c; D004926:Escherichia coli / Q000235:genetics*; D005998:Glycine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013053:Spectrophotometry; D013912:Threonine
D003574:Cytochrome c Group; C032139:iso-2-cytochrome C; D013912:Threonine; D045304:Cytochromes c; D011392:Proline; D005998:Glycine