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Synthetic and structural studies on Pyrularia pubera thionin: a single-residue mutation enhances activity against
Miquel Vila-Perelló1, Andrea Sánchez-Vallet, Francisco García-Olmedo
1Department of Experimental and Health Sciences, Pompeu Fabra University, Dr. Aiguader 80, E-08003 Barcelona, Spain.
Abstract:
The thionin from Pyrularia pubera (Pp-TH), a 47-residue peptide with four internal disulfide bonds, was efficiently produced by chemical synthesis. Its antimicrobial activity in vitro against several representative pathogens (EC(50)=0.3-3.0 microM) was identical to that of natural Pp-TH. This peptide has a unique Asp(32) instead of the consensus Arg found in other thionins of the same family. In order to evaluate the effect of this mutation, the Arg(32) analogue (Pp-TH(D32R)) was also synthesized and showed a significant increase in antibiotic activity against several Gram-negative bacteria, whereas it retained the same activity against other pathogens. The overall structure of Pp-TH(D32R) was maintained, though a slight decrease in the helical content of the peptide was observed.
Insights
Chemically synthesized Pyrularia pubera thionin (Pp-TH) showed antimicrobial activity. Mutating Asp32 to Arg32 (Pp-TH(D32R)) enhanced activity against Gram-negative bacteria, suggesting targeted antimicrobial peptide design.
Area of Science:
- Biochemistry
- Molecular Biology
- Antimicrobial Peptides
Background:
- Thionins are small, cysteine-rich proteins with diverse biological activities, including antimicrobial properties.
- Pyrularia pubera thionin (Pp-TH) is a 47-residue peptide characterized by four disulfide bonds and a unique Asp32 residue.
Purpose of the Study:
- To chemically synthesize Pp-TH and evaluate its antimicrobial activity.
- To investigate the functional impact of the Asp32 residue by creating and testing an Arg32 mutant (Pp-TH(D32R)).
Main Methods:
- Chemical synthesis of Pp-TH and its Asp32 to Arg32 mutant (Pp-TH(D32R)).
- In vitro antimicrobial activity assays against various bacterial pathogens.
- Structural analysis, including assessment of helical content.
Main Results:
- Synthesized Pp-TH exhibited antimicrobial activity comparable to the natural peptide.
- Pp-TH(D32R) demonstrated significantly enhanced antibiotic activity against Gram-negative bacteria.
- The overall structure of Pp-TH(D32R) was preserved, with only a minor reduction in helical content.
Conclusions:
- Chemical synthesis is an effective method for producing Pp-TH.
- The Asp32 residue plays a crucial role in modulating the antimicrobial spectrum of Pp-TH, particularly against Gram-negative bacteria.
- Targeted modifications of thionin residues can optimize their antimicrobial efficacy, offering potential for novel therapeutic development.