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Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states.
Biochimie. 2007 Nov; 89(11):1416-24.B

Abstract

Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state.

Authors+Show Affiliations

Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, UP, India.No affiliation info availableNo 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

17658212

Citation

Nallamsetty, Sreedevi, et al. "Accumulation of Partly Folded States in the Equilibrium Unfolding of Ervatamin A: Spectroscopic Description of the Native, Intermediate, and Unfolded States." Biochimie, vol. 89, no. 11, 2007, pp. 1416-24.
Nallamsetty S, Dubey VK, Pande M, et al. Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states. Biochimie. 2007;89(11):1416-24.
Nallamsetty, S., Dubey, V. K., Pande, M., Ambasht, P. K., & Jagannadham, M. V. (2007). Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states. Biochimie, 89(11), 1416-24.
Nallamsetty S, et al. Accumulation of Partly Folded States in the Equilibrium Unfolding of Ervatamin A: Spectroscopic Description of the Native, Intermediate, and Unfolded States. Biochimie. 2007;89(11):1416-24. PubMed PMID: 17658212.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states. AU - Nallamsetty,Sreedevi, AU - Dubey,Vikash K, AU - Pande,Monu, AU - Ambasht,P K, AU - Jagannadham,M V, Y1 - 2007/06/08/ PY - 2007/01/26/received PY - 2007/06/01/accepted PY - 2007/7/31/pubmed PY - 2008/1/24/medline PY - 2007/7/31/entrez SP - 1416 EP - 24 JF - Biochimie JO - Biochimie VL - 89 IS - 11 N2 - Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state. SN - 0300-9084 UR - https://www.unboundmedicine.com/medline/citation/17658212/Accumulation_of_partly_folded_states_in_the_equilibrium_unfolding_of_ervatamin_A:_spectroscopic_description_of_the_native_intermediate_and_unfolded_states_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0300-9084(07)00151-4 DB - PRIME DP - Unbound Medicine ER -