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Effect of Organic Solvents on the Activity, Stability and Secondary Structure of asclepain cI, Using FTIR and Molecular Dynamics Simulations

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Abstract

The present study aims at understanding the effect of organic solvents on the specific proteolytic activity and operational stability of asclepain cI in aqueous-organic media, using correlations between geometrical and structural parameters of asclepain cI. These correlations were determined by molecular dynamics (MD) simulations and the secondary structure of the enzyme validated by Fourier-transform Infrared (FTIR) spectroscopy. Asclepain cI exhibited significantly higher catalytic potential in 29 of the 42 aqueous-organic media tested, composed by 0.1 mM TRIS hydrochloride buffer pH 8 (TCB) and an organic solvent, than in buffer alone. Asclepain cI in water-organic miscible systems showed high FTIR spectral similarity with that obtained in TCB, while in immiscible systems the enzyme acquired different secondary structures than in buffer. Among the conditions studied, asclepain cI showed the highest catalytic potential in 50% v/v ethyl acetate in TCB. According to MD simulations, that medium elicited solvation and flexibility changes around the active center of asclepain cI and conducted to a new secondary structure with the active center preserved. These results provide valuable insights into the elucidation of the molecular mechanism of asclepain cI tolerance to organic solvents and pave the way for its future application for the synthesis of peptides in aqueous-organic media.

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Acknowledgements

The authors thank Dr. Sandra Signorella at IQUIR-CONICET-Rosario by the FTIR facilities and Dr. Estela Blaisten-Barojas that kindly provided the ethyl ester of acetic acid force field parameters. Anabella Lucía Origone is postdoctoral scholar at the National Council of Scientific and Technique Research (CONICET), Argentina. Esteban Vega Hissi and Sonia Barberis are scientific researchers at CCT-San Luis-CONICET, San Luis, Argentina.

Funding

This work was supported by the National University of San Luis, San Luis, Argentina (Grant Number 2-0718, 2018-2022).

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Authors and Affiliations

  1. Laboratory of Bromatology, Faculty of Chemistry, Biochemistry and Pharmacy (FQByF), National University of San Luis, Chacabuco N° 917, San Luis, Argentina

    Anabella L. Origone & Sonia E. Barberis

  2. Institute of Applied Physics (INFAP) – Technological Scientific Center of San Luis - National Council of Scientific and Technique Research (CONICET), Ejército de los Andes N° 950, Block II, 2nd Floor, 5700, San Luis, Argentina

    Anabella L. Origone & Sonia E. Barberis

  3. Physical-Chemistry Area, FQByF, National University of San Luis, Ejército de los Andes 950, 5700, San Luis, Argentina

    Esteban G. Vega Hissi

  4. Multidisciplinary Institute of Biological Research (IMIBIO) – CONICET, Ejército de los Andes N° 950, 5700, San Luis, Argentina

    Esteban G. Vega Hissi

  5. Plant Protein Research Center (CIProVe), National University of La Plata, Calle 47 y 115, La Plata, Argentina

    Constanza S. Liggieri

  6. Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario, Suipacha N° 531, Rosario, Santa Fe, Argentina

    Gerardo E. Camí

  7. Faculty of Engineering and Chemistry, Av. Pellegrini N° 3314, Rosario, Santa Fe, Argentina

    Gerardo E. Camí

  8. School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil N° 2085, Valparaiso, Chile

    Andrés Illanes

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  1. Anabella L. Origone
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Contributions

SB designed the experiments, did the data analysis together with AO, and wrote the manuscript. AO did the experimental trials, data collection and analysis. EVH performed molecular dynamics simulations. GC did the FTIR spectra. CL prepared the purified enzyme extracts. AI collaborated with SB in reviewing the final version of the manuscript. All authors contributed to the article and approved the submitted version.

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Correspondence to Esteban G. Vega Hissi or Sonia E. Barberis.

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Origone, A.L., Hissi, E.G.V., Liggieri, C.S. et al. Effect of Organic Solvents on the Activity, Stability and Secondary Structure of asclepain cI, Using FTIR and Molecular Dynamics Simulations. Protein J (2024). https://doi.org/10.1007/s10930-024-10182-4

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