Equilibrium unfolding of <Emphasis Type="Italic">A. niger</Emphasis> RNase: pH dependence of chemical and thermal denaturation |
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Authors: | Gundampati Ravi Kumar Anurag Sharma Moni Kumari Medicherla V Jagannadham Mira Debnath |
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Institution: | (1) School of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi, 221005, India;(2) Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India |
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Abstract: | Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH
using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions,
pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS
binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the
denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that
the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral
conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding
of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein,
suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition
from one molten globule like state (AMG1) into another (IMG2). |
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