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Expression of linear permutated variants from circular enterocin AS-48
Manuel Montalbán-López1, Manuel Martínez-Bueno, Eva Valdivia
1Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, C/Fuentenueva s/n, Granada, Spain.
Biochimie
|December 7, 2010
Summary
Circularization is crucial for bacteriocin AS-48 stability and activity. Permuted linear forms highlight the importance of the circular backbone and leader peptide for protein structure and function.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Bacteriocin AS-48 is a circular antimicrobial peptide.
- Understanding the role of its structure in stability and activity is essential.
Purpose of the Study:
- To investigate the role of head-to-tail circularization in bacteriocin AS-48 stability and activity.
- To construct and analyze permutated linear variants of AS-48.
Main Methods:
- Circular permutation was used to create two linear structural as-48A genes.
- Proteins were expressed in Escherichia coli and analyzed for stability and activity.
- Hybrid tagged proteins were generated and characterized.
Main Results:
- Leaderless linear AS-48 variants were absent in E. coli, supporting the importance of the circular backbone and leader peptide.
- Permutation and fusion strategies prevented cellular proteolysis, leading to inclusion body formation.
- Active hybrid tagged proteins were identified, with conserved hydrophobic/hydrophilic surfaces contributing to antibacterial activity.
- Opening the AS-48 molecule at G(23)-W(24) enhanced stability, indicating sequence and free amino acid criticality.
Conclusions:
- The circular structure of AS-48 is vital for its stability and likely its activity.
- Leader peptides play a significant role in protein stability and function.
- Specific sequence elements and free amino acids are critical for designing novel AS-48 variants with enhanced properties.
