Difference between revisions of "Part:BBa K4143336"

Latest revision as of 15:03, 11 October 2022

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 it has a minimum inhibitory concentration of 5 μM (E. coli C43(DE3))

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:

Figure 1: AlphaFold structural predictions for HBCM2 + targeting peptide

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.

Figure 2: Graphs of the root mean square fluctuation (RMSF) and root mean square deviation (RMSD) of the antimicrobial peptide. Given the low overall values (<2nm), it seems that this peptide is relatively stable in solvent. The RMSD graph is interpolated by a factor of 10 to reduce noise.

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).

Figure 3: SDS-PAGE gel of AMP (HBCM2) + encapsulin. Purified AMP is shown at the bottom of the gel.

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

3. Ceremuga M, Stela M, Janik E, Gorniak L, Synowiec E, Sliwinski T, Sitarek P, Saluk-Bijak J, Bijak M. Melittin-A Natural Peptide from Bee Venom Which Induces Apoptosis in Human Leukaemia Cells. Biomolecules. 2020 Feb 6;10(2):247. doi: 10.3390/biom10020247. PMID: 32041197; PMCID: PMC7072249.

4. Huan, Y., Kong, Q., Mou, H., & Yi, H. (2020). Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Frontiers in microbiology, 11, 582779. https://doi.org/10.3389/fmicb.2020.582779References

Sequence and Features