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Functional Stability and Structural Transitions of a Kunitz trypsin Inhibitor from Chickpea (CaTI2)

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Abstract

Enzymes are important tools for various applications. We have studied structural transitions and functional stability of a Kunitz trypsin inhibitor from Chickpea (CaTI2), a potent insect gut-protease inhibitor, under different stress conditions like non-neutral pH, elevated temperature and co-solvent concentrations. CaTI2 was cloned and expressed in an eukaryotic system P. pastoris and was investigated for conformational transitions using circular dichroism spectroscopy, differential scanning fluorimetry and activity assay. Native CaTI2 has a sheet dominant structure with 40% β sheets and possess a single tryptophan residue situated in the hydrophobic core of the enzyme. The recombinant inhibitor maintained its maximum activity under alkaline pH with its secondary structure intact between pH 6–10. CaTI2 was observed to be thermally stable up to 55 °C with a Tm of 61.3 °C above which the protein unfolds. On treating with chemical denaturant (urea), the CaTI2 lost its inhibitory potential and native conformation beyond 2 M urea concentration. Moreover, the protein unfolded at lower temperatures as the concentration of denaturant increased, suggesting more complex structural changes. Further, the stability of the inhibitor was found to be directly proportional to the solvent polarity. The data, herein offers significant information of inhibitor stability and activity which could be exploited for its further development into an effective pesticide.

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Abbreviations

BApNA:

Nα-Benzoyl-L-arginine para-nitroanilide

BPT:

Bovine pancreatic trypsin

CaTI2:

Cicer arietinum L Trypsin inhibitor 2

CD:

Circular dichroism

DSF:

Differential scanning fluorimetry

KTI:

Kunitz type inhibitor

PI:

Protease inhibitor

STI:

Soybean trypsin inhibitor

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Acknowledgements

ADB and ES thank University Grant Commission, India for Research Fellowship. SKR thanks Department of Science and Technology, India for Ramanujan Fellowship. Additional thanks to Dr. Sivaramaiah Nallapeta and Dr. Saji Menon (NanoTemper Technologies, India) for providing DSF facilities. Authors thank Dr. C. G. Suresh, Dr. Dhanasekaran Shanmugam and Ms. Sanskruthi Agarwal for valuable suggestions.

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Authors and Affiliations

  1. Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, 411008, India

    Ameya D. Bendre, Ekta Shukla & Sureshkumar Ramasamy

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    Ameya D. Bendre, Ekta Shukla & Sureshkumar Ramasamy

  3. National Centre for Cell Science, Ganeshkhind, Pune, 411007, India

    Ameya D. Bendre & Ekta Shukla

Authors
  1. Ameya D. Bendre
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  2. Ekta Shukla
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  3. Sureshkumar Ramasamy
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All experiments were performed by ADB and ES and were designed and monitored by SKR. All authors have approved the final article and submission.

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Correspondence to Ameya D. Bendre.

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Bendre, A.D., Shukla, E. & Ramasamy, S. Functional Stability and Structural Transitions of a Kunitz trypsin Inhibitor from Chickpea (CaTI2). Protein J 39, 350–357 (2020). https://doi.org/10.1007/s10930-020-09911-2

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