Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy |
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Authors: | Luciano Neves de Medeiros Renata Angeli Carolina G Sarzedas Ana Paula Valente Eleonora Kurtenbach |
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Institution: | a Instituto de Biofísica Carlos Chagas Filho, Programa de Biologia Molecular e Estrutural, Universidade Federal do Rio de Janeiro, Brazil b Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Brazil c Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Cidade Universitária, 21941-900, Rio de Janeiro RJ, Brazil |
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Abstract: | Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 15N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH. |
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Keywords: | CMH monohexosylceramide CSP chemical shift perturbation DmAMP Dahlia merckii antimicrobial peptide DPC dodecylphosphocholine Het-NOE Heteronuclear 15N Nuclear Overhauser Enhancement -M(IP)2C) mannosyldiinositolphosphoryl-ceramide pepLoop1 Gly12 to Ser19 Psd Pisum sativum defensin RsAFP1 Raphanus sativus antifungal peptide PC l-α-phosphatidylcholine" target="_blank">l-α-phosphatidylcholine PRE paramagnetic relaxation enhancement R1 heteronuclear longitudinal relaxation time R2 heteronuclear transverse relaxation time |
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