Hard nut to crack: Solving the disulfide linkage pattern of the Neosartorya (Aspergillus) fischeri antifungal protein 2

Váradi, Györgyi and Kele, Zoltán and Czajlik, András and Borics, Attila and Bende, Gábor and Papp, Csaba Gergő and Rákhely, Gábor and Tóth, Gábor and Batta, Gyula and Galgóczi, László Norbert (2023) Hard nut to crack: Solving the disulfide linkage pattern of the Neosartorya (Aspergillus) fischeri antifungal protein 2. PROTEIN SCIENCE, 32 (7). No-e4692. ISSN 0961-8368

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As a consequence of the fast resistance spreading, a limited number of drugs are available to treat fungal infections. Therefore, there is an urgent need to develop new antifungal treatment strategies. The features of a disulfide bond-stabilized antifungal protein, NFAP2 secreted by the mold Neosartorya (Aspergillus) fischeri render it to be a promising template for future protein-based antifungal drug design, which requires knowledge about the native disulfide linkage pattern as it is one of the prerequisites for biological activity. However, in the lack of tryptic and chymotryptic proteolytic sites in the ACNCPNNCK sequence, the determination of the disulfide linkage pattern of NFAP2 is not easy with traditional mass spectrometry-based methods. According to in silico predictions working with a preliminary nuclear magnetic resonance (NMR) solution structure, two disulfide isomers of NFAP2 (abbacc and abbcac) were possible. Both were chemically synthesized; and comparative reversed-phase high-performance liquid chromatography, electronic circular dichroism and NMR spectroscopy analyses, and antifungal susceptibility and efficacy tests indicated that the abbcac is the native pattern. This knowledge allowed rational modification of NAFP2 to improve the antifungal efficacy and spectrum through the modulation of the evolutionarily conserved gamma-core region, which is responsible for the activity of several antimicrobial peptides. Disruption of the steric structure of NFAP2 upon gamma-core modification led to the conclusions that this motif may affect the formation of the biologically active three-dimensional structure, and that the gamma-core modulation is not an efficient tool to improve the antifungal efficacy or to change the antifungal spectrum of NFAP2.

Item Type: Article
Uncontrolled Keywords: antifungal protein, disulfide linkage pattern, drug design, protein structure, protein synthesis
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3015 Molecular biology / molekuláris biológia
Q Science / természettudomány > QR Microbiology / mikrobiológia
Depositing User: MTMT SWORD
Date Deposited: 26 Sep 2023 13:57
Last Modified: 26 Sep 2023 13:57

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