title: Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c.
abstract: It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more stable than the PMG state by 5.4 ± 0.1 kcal mol(-1), whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol(-1). A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states.
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
authors.forename: Md Khurshid
authors.initials: MK
authors.lastname: Alam Khan
authors.forename: Md Hamidur
authors.initials: MH
authors.lastname: Rahaman
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
authors.forename: Tej P
authors.initials: TP
authors.lastname: Singh
authors.forename: Ali A
authors.initials: AA
authors.lastname: Moosavi-Movahedi
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
mesh_terms: D000818:Animals; D045304:Cytochromes c / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006736:Horses; D018021:Lithium Chloride / Q000494:pharmacology; D008968:Molecular Conformation; D011489:Protein Denaturation / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects; D013816:Thermodynamics
chemical_list: D045304:Cytochromes c; D018021:Lithium Chloride