title: Alcohol and temperature induced conformational transitions in ervatamin B: sequential unfolding of domains.
abstract: The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly alpha-helical to beta-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the alpha-helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.
authors.affiliation: Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India.
authors.forename: Suman
authors.initials: S
authors.lastname: Kundu
authors.forename: Monica
authors.initials: M
authors.lastname: Sundd
authors.forename: Medicherla V
authors.initials: MV
authors.lastname: Jagannadham
mesh_terms: D000438:Alcohols; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000438:Alcohols; D003546:Cysteine Endopeptidases; C406685:ervatamin B; D000432:Methanol