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Effect of alkyl alcohols on partially unfolded state of proteinase K: Differential stability of alpha-helix and beta-sheet rich regions of the enzyme.
Biochimie. 2009 Aug; 91(8):951-60.B

Abstract

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to alpha/beta class of proteins and maintains structural integrity in the range of pH 7.0-3.0. Enzyme acquires partially unfolded conformation (U(P)) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from alpha/beta to beta-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U(P) state occurred in two steps with the formation of beta state at low alcohol concentration followed by stabilization of beta state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.

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Authors+Show Affiliations

Tomar R
Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
Dubey VK
No affiliation info available
Jagannadham MV
No affiliation info available

MeSH

2-PropanolAlcoholsAscomycotaCircular DichroismDose-Response Relationship, DrugEndopeptidase KEnzyme StabilityFluorescent DyesGuanidineHydrogen-Ion ConcentrationProtein DenaturationProtein FoldingProtein Structure, SecondaryProtein Structure, TertiarySpectrum AnalysisTemperatureWater

Pub Type(s)

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

Language

eng

PubMed ID

19403104

Citation

Tomar, Ritu, et al. "Effect of Alkyl Alcohols On Partially Unfolded State of Proteinase K: Differential Stability of Alpha-helix and Beta-sheet Rich Regions of the Enzyme." Biochimie, vol. 91, no. 8, 2009, pp. 951-60.
Tomar R, Dubey VK, Jagannadham MV. Effect of alkyl alcohols on partially unfolded state of proteinase K: Differential stability of alpha-helix and beta-sheet rich regions of the enzyme. Biochimie. 2009;91(8):951-60.
Tomar, R., Dubey, V. K., & Jagannadham, M. V. (2009). Effect of alkyl alcohols on partially unfolded state of proteinase K: Differential stability of alpha-helix and beta-sheet rich regions of the enzyme. Biochimie, 91(8), 951-60. https://doi.org/10.1016/j.biochi.2009.04.013
Tomar R, Dubey VK, Jagannadham MV. Effect of Alkyl Alcohols On Partially Unfolded State of Proteinase K: Differential Stability of Alpha-helix and Beta-sheet Rich Regions of the Enzyme. Biochimie. 2009;91(8):951-60. PubMed PMID: 19403104.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Effect of alkyl alcohols on partially unfolded state of proteinase K: Differential stability of alpha-helix and beta-sheet rich regions of the enzyme. AU - Tomar,Ritu, AU - Dubey,Vikash Kumar, AU - Jagannadham,M V, Y1 - 2009/05/03/ PY - 2008/10/05/received PY - 2009/04/16/accepted PY - 2009/5/1/entrez PY - 2009/5/1/pubmed PY - 2009/10/14/medline SP - 951 EP - 60 JF - Biochimie JO - Biochimie VL - 91 IS - 8 N2 - Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to alpha/beta class of proteins and maintains structural integrity in the range of pH 7.0-3.0. Enzyme acquires partially unfolded conformation (U(P)) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from alpha/beta to beta-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U(P) state occurred in two steps with the formation of beta state at low alcohol concentration followed by stabilization of beta state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K. SN - 1638-6183 UR - https://www.unboundmedicine.com/medline/citation/19403104/Effect_of_alkyl_alcohols_on_partially_unfolded_state_of_proteinase_K:_Differential_stability_of_alpha_helix_and_beta_sheet_rich_regions_of_the_enzyme_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0300-9084(09)00113-8 DB - PRIME DP - Unbound Medicine ER -