TY  - JOUR
AU  - Marković, Srdjan
AU  - Andrejević, Natalija S.
AU  - Milošević, Jelica
AU  - Polović, Natalija
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6289
AB  - The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group’s reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific β-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal β-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases.
PB  - MDPI
T2  - Biomimetics
T1  - Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development
VL  - 8
IS  - 3
SP  - 281
DO  - 10.3390/biomimetics8030281
ER  - 

@article{
author = "Marković, Srdjan and Andrejević, Natalija S. and Milošević, Jelica and Polović, Natalija",
year = "2023",
abstract = "The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group’s reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific β-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal β-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases.",
publisher = "MDPI",
journal = "Biomimetics",
title = "Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development",
volume = "8",
number = "3",
pages = "281",
doi = "10.3390/biomimetics8030281"
}

Marković, S., Andrejević, N. S., Milošević, J.,& Polović, N.. (2023). Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development. in Biomimetics
MDPI., 8(3), 281.
https://doi.org/10.3390/biomimetics8030281

Marković S, Andrejević NS, Milošević J, Polović N. Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development. in Biomimetics. 2023;8(3):281.
doi:10.3390/biomimetics8030281 .

Marković, Srdjan, Andrejević, Natalija S., Milošević, Jelica, Polović, Natalija, "Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development" in Biomimetics, 8, no. 3 (2023):281,
https://doi.org/10.3390/biomimetics8030281 . .