Best Improved Part
The original sequence of our MaSAMP can be found on NCBI database (https://www.ncbi.nlm.nih.gov/protein/KDO72074.1), discovered by Huang et al., 2021 [1]. As described in our other page, this antimicrobial peptide (AMP) consists of 67 amino acids including a double helix hairpin. Huang et al., 2021 pointed out the main function part was the second alpha helix. We decided to use the original second alpha helix as an existing part. To increase the efficiency, we chemically synthesized mutants of the second alpha helix produced by our software to test whether the improved part functions better than the older part. We synthesized 6 candidates and test the potential sterilizing effect via fluorescent staining and measuring OD600.
The improvement was supported by both experimental measurement and modeled measurements. The experimental measurement was guided by our software. The software provided not only the amino acid sequence of mutants but also biophysical parameters as a reference. Since lyticity did not change, we may not need to worry about lysing the blood cells. Also, the total hydrophobicity does not change much. So, we used total charge as our first guidance to screen the mutants. The graph below shows the Results of single and double substitution by lysine with biophysical parameters (lyticity, total hydrophobicity, net charge and 3D hydrophobic Moment Vector. The improved 6 mutants with higher net charge were on the bottom. That is the modelling support. For further information, please check Software page.
Figure 1. Results of single and double substitution by lysine with biophysical parameters (lyticity, total hydrophobicity, net charge and 3D hydrophobic Moment Vector. Red characters point out the position substituted. Blue characters are the main function domain (second alpha helix). The following biophysical parameters are calculated via our assembled toolkit – ProteinOpti.
We chemically synthesized it for testing. The table below shows the result after treating with 5 hours from the original concentration of OD600 = 0.4.
The SAMP_4 and SAMP_6 showed improved sterilizing effect, which supports our software. SAMP_6 is the best improved part (BBa_K4587224). Also, our screening showed positions suitable for substituting with lysine (K). The wetlab result shows that our software can point out the direction and guide the downstream wetlab experiments.