Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c.
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.
Alam Khan
Md Khurshid
MK
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
Rahaman
Md Hamidur
MH
Hassan
Md Imtaiyaz
MI
Singh
Tej P
TP
Moosavi-Movahedi
Ali A
AA
Ahmad
Faizan
F
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
D045304:Cytochromes c; D018021:Lithium Chloride