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Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants.
Biochemistry. 2003 Feb 18; 42(6):1684-95.B

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.

Authors+Show Affiliations

Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi - 110 025, India.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12578383

Citation

Qureshi, Shabir H., et al. "Conformational and Thermodynamic Characterization of the Molten Globule State Occurring During Unfolding of Cytochromes-c By Weak Salt Denaturants." Biochemistry, vol. 42, no. 6, 2003, pp. 1684-95.
Qureshi SH, Moza B, Yadav S, et al. Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants. Biochemistry. 2003;42(6):1684-95.
Qureshi, S. H., 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), 1684-95.
Qureshi SH, et al. Conformational and Thermodynamic Characterization of the Molten Globule State Occurring During Unfolding of Cytochromes-c By Weak Salt Denaturants. Biochemistry. 2003 Feb 18;42(6):1684-95. PubMed PMID: 12578383.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants. AU - Qureshi,Shabir H, AU - Moza,Beenu, AU - Yadav,Sushma, AU - Ahmad,Faizan, PY - 2003/2/13/pubmed PY - 2003/5/3/medline PY - 2003/2/13/entrez SP - 1684 EP - 95 JF - Biochemistry JO - Biochemistry VL - 42 IS - 6 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/12578383/Conformational_and_thermodynamic_characterization_of_the_molten_globule_state_occurring_during_unfolding_of_cytochromes_c_by_weak_salt_denaturants_ DB - PRIME DP - Unbound Medicine ER -