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Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding.
J Biotechnol. 2007 Sep 30; 131(4):404-17.JB

Abstract

pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway.

Authors+Show Affiliations

Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 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

17825936

Citation

Pande, Monu, et al. "Conformational Plasticity of Cryptolepain: Accumulation of Partially Unfolded States in Denaturants Induced Equilibrium Unfolding." Journal of Biotechnology, vol. 131, no. 4, 2007, pp. 404-17.
Pande M, Dubey VK, Sahu V, et al. Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding. J Biotechnol. 2007;131(4):404-17.
Pande, M., Dubey, V. K., Sahu, V., & Jagannadham, M. V. (2007). Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding. Journal of Biotechnology, 131(4), 404-17.
Pande M, et al. Conformational Plasticity of Cryptolepain: Accumulation of Partially Unfolded States in Denaturants Induced Equilibrium Unfolding. J Biotechnol. 2007 Sep 30;131(4):404-17. PubMed PMID: 17825936.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding. AU - Pande,Monu, AU - Dubey,Vikash K, AU - Sahu,Vishal, AU - Jagannadham,Medicherla V, Y1 - 2007/08/07/ PY - 2007/04/02/received PY - 2007/07/25/revised PY - 2007/08/01/accepted PY - 2007/9/11/pubmed PY - 2007/12/15/medline PY - 2007/9/11/entrez SP - 404 EP - 17 JF - Journal of biotechnology JO - J Biotechnol VL - 131 IS - 4 N2 - pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway. SN - 0168-1656 UR - https://www.unboundmedicine.com/medline/citation/17825936/Conformational_plasticity_of_cryptolepain:_accumulation_of_partially_unfolded_states_in_denaturants_induced_equilibrium_unfolding_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0168-1656(07)01504-0 DB - PRIME DP - Unbound Medicine ER -