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Structural Stability and Unfolding Properties of Cutinases from Thermobifida fusca

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Abstract

A comparative analysis of the structural and functional aspects along with equilibrium unfolding of two homologous cutinases, Cut1 and Cut2, from Thermobifida fusca was carried out. The CD and fluorescence profile at different pH in the range of 6 to 9 showed no structural variations for both cutinases, indicating their stability to a wide range of pH. Tryptophan quenching studies suggested that all the four Trp residues in the protein are in inaccessible hydrophobic pockets. Further, near-UV CD analysis of tertiary structure revealed a dissimilar distribution of aromatic amino acid on the surface of these two enzymes. Denaturation profiles obtained in aqueous solutions of the guanidine hydrochloride revealed different tolerance levels for unfolding of the two cutinases, with Cut2 showing higher resistivity to unfolding in comparison to Cut1. Both cutinases retained all the structural parameters even in the presence of 8 M urea, indicating the protein to be highly resistant to urea-induced unfolding. Structural study by homology modeling revealed a high resemblance of secondary structure between the two cutinases; however, their tertiary structure, hydrophobicity, and surface electrostatic properties were very different, which contributed to the difference in the structural stability of these two cutinases.

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Acknowledgments

The authors acknowledge a financial support from DST through project for carrying out the experiments, Prof. Sauman Basak, SINP, Kolkata, and DBT for sponsored instrumentation facility, IIT Guwahati, for providing CD spectroscopy facility, Ms. Maupriya SINP, Kolkata, and Dr. Mohitosh, IIT Guwahati, for their assistance in carrying out CD spectroscopy, and Ms. Debomitra, IIT Guwahati, for her valuable suggestions in carrying out structural homology modeling of protein.

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  1. Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India

    Krishnamoorthy Hegde & Veeranki Venkata Dasu

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  1. Krishnamoorthy Hegde
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  2. Veeranki Venkata Dasu
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Correspondence to Veeranki Venkata Dasu.

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Hegde, K., Dasu, V.V. Structural Stability and Unfolding Properties of Cutinases from Thermobifida fusca . Appl Biochem Biotechnol 174, 803–819 (2014). https://doi.org/10.1007/s12010-014-1037-5

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