Part:BBa_K4143336
Antimicrobial Pepide: HBCM2
Antimicrobial peptides, or AMPs, are naturally occurring peptides used by organisms to combat pathogenic infections.They have recently gained traction as an alternative to antibiotics due to the rise of antimicrobial-resistant infections. HBCM2, an α-helical AMP hybrid of cecropin and melittin peptides, is particularly active against gram-negative bacteria. Its target is membrane permeabilization and has a Minimum Inhibitory Concentration of 5 μM (E. coli C43(DE3))
Contents
Usage and Biology
AlphaFold Structural Characterization
Because no structural information about HBCM2 was available, we generated predictions of the AMP + targeting peptide using AlphaFold due to its purported ability to predict nearly accurate protein structures based only on primary sequences. It predicted a primarily alpha-helical structure. This structure is shown below:
Molecular Dynamic Stability Analysis
The first step to understanding the stability of the HBMC2 antimicrobial peptide was to run it in a 10ns conventional molecular dynamics simulation. On the most superficial level, when considering RMSD and RMSF, it seemed that the peptide was mostly stable in its conformation. The RMSD and RMSF belows always remained within 2nm throughout the course of the simulation, although there were periods of time where major structural events seemed to occur, as well as amino acids with slightly higher levels of fluctuation than others.
The RMSF graph suggested that the areas of the protein with the highest comparative fluctuation were the N-terminus and C-terminus, which AlphaFold determined to be composed of mostly ambiguous secondary structure (neither alpha-helix nor beta-sheet). Since this area takes up more than half of the peptide, it is noteworthy, since peptide termini are generally the most flexible region of any form of amino acid chain.
Visualizations of Trial 1 (the simulation with the highest RMSD and RMSF) confirmed this observation, but they also demonstrated an unexpected flexibility within the alpha-helix chain that makes up the rest of the peptide. Hydrophobic interactions between the C-terminus and residues Thr20 and Thr21 lead to a bending of the alpha-helix chain.
Although previous studies have suggested that the HBCM2 peptide binds well with E. coli O111:B4 lipopolysaccharide,[2] this result suggests a possible sensitivity to environmental conditions. Useful further studies might be to see (1) if such structural change is reversible, (2) how the peptide reacts to the encapsulin environment, and (3) which conformations of the antimicrobial peptide can stably bind to the LPS and inhibit E. coli growth.
AMP Purification
After ordering our part from IDT, we expressed it in BL21 E. coli and purified it along with an encapsulin (see BBa_K4143337 for the encapsulin, BBa_K4143340 for the composite part). An SDS-PAGE gel confirmed the presence of HBCM2 within our encapsulin (Figure 3).
References
1. Lee, T. H., Carpenter, T. S., D'haeseleer, P., Savage, D. F., & Yung, M. C. (2020). Encapsulin carrier proteins for enhanced expression of antimicrobial peptides. Biotechnology and bioengineering, 117(3), 603–613. https://doi.org/10.1002/bit.27222
2. Scott, M G et al. “Biological properties of structurally related alpha-helical cationic antimicrobial peptides.” Infection and immunity vol. 67,4 (1999): 2005-9. doi:10.1128/IAI.67.4.2005-2009.1999
Sequence and Features
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