Mutation of asparagine 52 to glycine promotes the alkaline form of iso-1-cytochrome c and causes loss of cooperativity in acid unfolding.
The kinetics and thermodynamics of the alkaline and acid conformational transitions of a Lys 79 --> Ala/Asn 52 --> Gly (A79G52) variant of iso-1-cytochrome c are studied. The Lys 79 --> Ala mutation is designed to limit heme ligation in the alkaline conformer to Lys 73. The Asn 52 --> Gly mutation is intended to shift the population of the alkaline conformer to physiological pH based on the hierarchical nature of the cooperative substructures of this protein. The midpoint pH for formation of the alkaline conformer is approximately 7.45. The kinetics for the alkaline conformational transition of the A79G52 variant are consistent with the ionization constant, pK(H), for the trigger group controlling formation of the alkaline conformer being approximately 9.5. This pK(H) is low for alkaline conformers involving lysine-heme ligation but is consistent with the pK(a) of the highest of three ionizable groups which modulate formation of the histidine-heme alkaline conformer of a His 73 variant of iso-1-cytochrome c [Martinez, R. E., and Bowler, B. E. (2004) J. Am. Chem. Soc. 126, 6751-6758]. The acid transition of the A79G52 variant is split into two phases. Both the Lys 79 --> Ala and Asn 52 --> Gly mutations are expected to affect the buried hydrogen bond network of cytochrome c, suggesting that this network is an important modulator of the acid unfolding of cytochrome c.
Department of Chemistry and Biochemistry, University of Denver, 2190 East Iliff Avenue, Denver, Colorado 80208-2436, USA.
Saritha
S
Baddam
Bruce E
BE
Bowler
D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D045304:Cytochromes c / Q000235:genetics* / Q000302:isolation & purification; D005998:Glycine / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification; D013816:Thermodynamics
C014059:CYC1 protein, S cerevisiae; D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D001216:Asparagine; D045304:Cytochromes c; D005998:Glycine