A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0.
Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8-naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25 degrees C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural characteristics of a pre-molten-globule state at pH 2 and 25 degrees C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28 degrees C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein.
Alam  Khan
Md Khurshid
MK
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India.
Das
Utpal
U
Rahaman
Md Hamidur
MH
Hassan
Md Imtaiyaz
MI
Srinivasan
A
A
Singh
Tej P
TP
Ahmad
Faizan
F
D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics*; D004795:Enzyme Stability; D006736:Horses / Q000235:genetics* / Q000378:metabolism; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature
D003574:Cytochrome c Group