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Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants.
Abstract: The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation X (intermediate) conformation and the second denaturation phase, X conformation D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation X conformation and X conformation D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation X conformation and X conformation D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.
Publication Date: 2003-02-13 PubMed ID: 12578383DOI: 10.1021/bi0271042Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research investigates the denaturation of bovine and horse cytochromes-c by weak salt denaturants and characterizes the change in conformation and thermal properties of the molecules. The results indicate that one stage of change, identified as the X state, is a molten globule state, and that the native state is more stable than the X state.
Denaturation of Cytochromes-c by Weak Salt Denaturants
- Through the use of weak salt denaturants LiCl and CaCl2, the research focused on the denaturation, or alteration, of bovine and horse cytochromes-c proteins at 25 degrees Celsius.
- Changes in molar absorbance and circular dichroism were monitored for the, leading to findings that the denaturation process is biphasic, or two-staged in terms of how cytochromes-c conformation alters.
Identification and Characterization of Intermediate States
- The measured observations revealed two phases of denaturation. The first phase shifted the native conformation (N) to an intermediate conformation state (X). The second phase transitioned from this intermediate state (X) to the denatured state (D). It was noted that these transitions are reversible.
- Various techniques, including far-UV CD, ANS binding, and intrinsic viscosity measurements, were used to characterize the intermediate X state. Through these methods, the researchers concluded that the X state is a “molten globule state”.
Stability of Different States and Effect of Temperature
- By analyzing the denaturation transition curves in terms of Gibbs energy change, the researchers found that the native state (N) is more stable than the molten globule state (X) by about 9.55 kcal per mol. The X state, on the other hand, is 1.40 kcal mol-1 more stable than the denatured state (D).
- The researchers also measured the impact of temperature on the N-X and X-D equilibria in the context of different denaturant concentrations. This analysis revealed that 30% of the hydrophobic groups in the molten globule state are not accessible to the water.
Cite This Article
APA
Qureshi SH, Moza B, Yadav S, Ahmad F.
(2003).
Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants.
Biochemistry, 42(6), 1684-1695.
https://doi.org/10.1021/bi0271042 Publication
Researcher Affiliations
- Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi - 110 025, India.
MeSH Terms
- Animals
- Calcium Chloride / chemistry
- Cattle
- Circular Dichroism
- Cytochrome c Group / chemistry
- Enzyme Stability
- Horses
- Hydrogen-Ion Concentration
- Hydrophobic and Hydrophilic Interactions
- Kinetics
- Lithium Chloride / chemistry
- Models, Chemical
- Protein Conformation
- Protein Denaturation
- Protein Folding
- Protein Structure, Secondary
- Salts / chemistry
- Temperature
- Thermodynamics
Citations
This article has been cited 10 times.- Parray ZA, Ahmad F, Chaudhary AA, Rudayni HA, Al-Zharani M, Hassan MI, Islam A. Size-Dependent Interplay of Volume Exclusion Versus Soft Interactions: Cytochrome c in Macromolecular Crowded Environment. Front Mol Biosci 2022;9:849683.
- Naiyer A, Khan B, Hussain A, Islam A, Alajmi MF, Hassan MI, Sundd M, Ahmad F. Stability of uniformly labeled ((13)C and (15)N) cytochrome c and its L94G mutant. Sci Rep 2021 Mar 24;11(1):6804.
- Parray ZA, Ahmad F, Alajmi MF, Hussain A, Hassan MI, Islam A. Interaction of polyethylene glycol with cytochrome c investigated via in vitro and in silico approaches. Sci Rep 2021 Mar 19;11(1):6475.
- Zuk PJ, Cichocki B, Szymczak P. GRPY: An Accurate Bead Method for Calculation of Hydrodynamic Properties of Rigid Biomacromolecules. Biophys J 2018 Sep 4;115(5):782-800.
- Rahaman H, Alam Khan MK, Hassan MI, Islam A, Moosavi-Movahedi AA, Ahmad F. Heterogeneity of equilibrium molten globule state of cytochrome c induced by weak salt denaturants under physiological condition. PLoS One 2015;10(4):e0120465.
- Zaidi S, Hassan MI, Islam A, Ahmad F. The role of key residues in structure, function, and stability of cytochrome-c. Cell Mol Life Sci 2014 Jan;71(2):229-55.
- Rahaman H, Zhou S, Dodia C, Feinstein SI, Huang S, Speicher D, Fisher AB. Increased phospholipase A2 activity with phosphorylation of peroxiredoxin 6 requires a conformational change in the protein. Biochemistry 2012 Jul 10;51(27):5521-30.
- Motta FN, Bastos IM, Faudry E, Ebel C, Lima MM, Neves D, Ragno M, Barbosa JA, de Freitas SM, Santana JM. The Trypanosoma cruzi virulence factor oligopeptidase B (OPBTc) assembles into an active and stable dimer. PLoS One 2012;7(1):e30431.
- Alam Khan MK, Rahaman MH, Hassan MI, Singh TP, Moosavi-Movahedi AA, Ahmad F. Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c. J Biol Inorg Chem 2010 Nov;15(8):1319-29.
- Alam Khan MK, Das U, Rahaman MH, Hassan MI, Srinivasan A, Singh TP, Ahmad F. A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0. J Biol Inorg Chem 2009 Jun;14(5):751-60.
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