Dataset Information

DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study.

ABSTRACT: PAF and related antifungal proteins are promising antimicrobial agents. They have highly stable folds around room temperature due to the presence of 3-4 disulfide bonds. However, unfolded states persist and contribute to the thermal equilibrium in aqueous solution, and low-populated states might influence their biological impact. To explore such equilibria during dimethyl sulfoxide (DMSO)-induced chemical unfolding, we studied PAF and its inactive variant PAF^D19S using nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). According to the NMR monitoring at 310 K, the folded structures disappear above 80 v/v% DMSO concentration, while the unfolding is completely reversible. Evaluation of a few resolved peaks from viscosity-compensated ¹⁵N-¹H HSQC spectra of PAF yielded ∆G = 23 ± 7 kJ/M as the average value for NMR unfolding enthalpy. The NMR-based structures of PAF and the mutant in 50 v/v% DMSO/H₂O mixtures were more similar in the mixed solvents then they were in water. The ¹⁵N NMR relaxation dynamics in the same mixtures verified the rigid backbones of the NMR-visible fractions of the proteins; still, enhanced dynamics around the termini and some loops were observed. DSC monitoring of the T_m melting point showed parabolic dependence on the DMSO molar fraction and suggested that PAF is more stable than the inactive PAF^D19S. The DSC experiments were irreversible due to the applied broad temperature range, but still suggestive of the endothermic unfolding of PAF.

SUBMITTER: Czajlik A

PROVIDER: S-EPMC9864379 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study.

Czajlik András A Batta Ágnes Á Kerner Kinga K Fizil Ádám Á Hajdu Dorottya D Raics Mária M Kövér Katalin E KE Batta Gyula G

International journal of molecular sciences 20230107 2

PAF and related antifungal proteins are promising antimicrobial agents. They have highly stable folds around room temperature due to the presence of 3-4 disulfide bonds. However, unfolded states persist and contribute to the thermal equilibrium in aqueous solution, and low-populated states might influence their biological impact. To explore such equilibria during dimethyl sulfoxide (DMSO)-induced chemical unfolding, we studied PAF and its inactive variant PAF<sup>D19S</sup> using nuclear magneti ...[more]

PMID: 36674720

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Project description:PAFs are short cationic and tryptophan-rich synthetic peptides with cell-penetrating antifungal activity. They show potent and selective killing activity against major fungal pathogens and low toxicity to other eukaryotic and bacterial cells. These properties make them a promising alternative to fulfill the need of novel antifungals with potential applications in crop protection, food preservation, and medical therapies. However, the difficulties of cost-effective manufacturing of PAFs by chemical synthesis or biotechnological production in microorganisms have hampered their development for practical use. This work explores the feasibility of using rice seeds as an economical and safe production system of PAFs. The rationally designed PAF102 peptide with improved antifungal properties was selected for assessing PAF biotechnological production. Two different strategies are evaluated: (1) the production as a single peptide targeted to protein bodies and (2) the production as an oleosin fusion protein targeted to oil bodies. Both strategies are designed to offer stability to the PAF peptide in the host plant and to facilitate its downstream purification. Our results demonstrate that PAF does not accumulate to detectable levels in rice seeds when produced as a single peptide, whereas it is successfully produced as fusion protein to the Oleosin18, up to 20 μg of peptide per gram of grain. We show that the expression of the chimeric Ole18-PAF102 gene driven by the Ole18 promoter results in the specific accumulation of the fusion protein in the embryo and aleurone layer of the rice seed. Ole18-PAF102 accumulation has no deleterious effects on seed yield, germination capacity, or seedling growth. We also show that the Oleosin18 protein serves as carrier to target the fusion protein to oil bodies facilitating PAF102 recovery. Importantly, the recovered PAF102 is active against the fungal phytopathogen Fusarium proliferatum. Altogether, our results prove that the oleosin fusion technology allows the production of PAF bioactive peptides to assist the exploitation of these antifungal compounds.

Dataset Information

DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study.

Publications

DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study.

Similar Datasets

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