Supplementary file 1_Experimental screening and structure-informed engineering of peptide-fused bacteriophage lysins with enhanced activity against Gram-negative bacteria.docx

The variants (Syn138, Pad138, and CecA138) showing enhanced activity in 150 mM NaCl solution and in sera were found to be fused with peptides, with most of their residues forming α-helical structures, as suggested by computational structure analysis using AlphaFold and PSIPRED. In contrast, the inactive variants were fused to peptides predicted to adopt non-helical structures and possess a relatively small number of positively charged residues.

This observation suggested that the presence of more α-helical structures within a positively charged fused peptide may be an important feature for designing effective lysin fusions under physiological conditions. This was further examined by fusing two selected peptides with another lysin, LysPd149. These findings suggest that protein structure prediction and charge analysis could provide a more efficient way to rationally select or design peptides to be fused with lysins against Gram-negative bacteria before protein expression, saving time and costs associated with the current trial-and-error fusion strategies.</p>