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Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates.
J Biochem Mol Biol. 2002 Mar 31; 35(2):143-54.JB

Abstract

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the alpha + beta class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0- 2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a beta-sheet conformation and shows a strong binding to 8-anilino-1- napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.

Authors+Show Affiliations

Sundd M
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India.
Kundu S
No affiliation info available
Jagannadham MV
No affiliation info available

MeSH

Anilino NaphthalenesulfonatesCircular DichroismCysteine EndopeptidasesGuanidineHydrogen-Ion ConcentrationProtein ConformationProtein DenaturationProtein Structure, SecondarySpectrometry, FluorescenceSpectrophotometryUrea

Pub Type(s)

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

Language

eng

PubMed ID

12297023

Citation

Sundd, Monica, et al. "Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates." Journal of Biochemistry and Molecular Biology, vol. 35, no. 2, 2002, pp. 143-54.
Sundd M, Kundu S, Jagannadham MV. Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates. J Biochem Mol Biol. 2002;35(2):143-54.
Sundd, M., Kundu, S., & Jagannadham, M. V. (2002). Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates. Journal of Biochemistry and Molecular Biology, 35(2), 143-54.
Sundd M, Kundu S, Jagannadham MV. Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates. J Biochem Mol Biol. 2002 Mar 31;35(2):143-54. PubMed PMID: 12297023.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates. AU - Sundd,Monica, AU - Kundu,Suman, AU - Jagannadham,Medicherla V, PY - 2002/9/26/pubmed PY - 2002/12/5/medline PY - 2002/9/26/entrez SP - 143 EP - 54 JF - Journal of biochemistry and molecular biology JO - J Biochem Mol Biol VL - 35 IS - 2 N2 - The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the alpha + beta class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0- 2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a beta-sheet conformation and shows a strong binding to 8-anilino-1- napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily. SN - 1225-8687 UR - https://www.unboundmedicine.com/medline/citation/12297023/Acid_and_chemical_induced_conformational_changes_of_ervatamin_B__Presence_of_partially_structured_multiple_intermediates_ DB - PRIME DP - Unbound Medicine ER -