title: Thermodynamic effects of the alteration of the axial ligand on the unfolding of thermostable cytochrome C.
abstract: The role the axial methionine plays in the conformational properties and thermostability of the heme active site has been investigated with the help of site-specific mutations at the axial Met69 position with His (M69H) and Ala (M69A) in thermostable cytochrome c(552) from Thermus thermophilus. Detailed circular dichroism, direct electrochemistry, and other spectroscopic studies have been employed to investigate the thermally induced and GdnHCl-induced unfolding properties of the heme active site of the wild type and the mutants of cytochrome c(552). We observed an unusually high thermodynamic and thermal stability of the M69A mutant compared to that of wild-type cytochrome c(552). However, the M69H mutant exhibited a slightly lower unfolding free energy compared to that of the wild-type protein. The high conformational stability of the M69A mutant was attributed to the presence of residual structure in the unfolded state as well as to the altered conformation in the folded state of this mutant of cytochrome c(552). This study thus supports the view that apart from the folded state, the unfolded state of a protein may also make a significant contribution to the stability of a protein.
authors: Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
authors: Rabindra Kumar
authors: RK
authors: Behera
authors: Hiroshi
authors: H
authors: Nakajima
authors: Jitumani
authors: J
authors: Rajbongshi
authors: Yoshihito
authors: Y
authors: Watanabe
authors: Shyamalava
authors: S
authors: Mazumdar
mesh_terms: D020134:Catalytic Domain / Q000187:drug effects; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D055664:Electrochemical Techniques; D019791:Guanidine / Q000494:pharmacology; D006418:Heme / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D003574:Cytochrome c Group; D006418:Heme; C022302:cytochrome C-552; D019791:Guanidine