Conclusion:Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides. General significance:The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipatic, since this feature seems to help the toroidal pores formation process. Highlights A new Temporin B analogue, TB_KKG6A, was designed.TB_KKG6A is active against Gram-positive and Gram-negative bacteria.The peptide strongly interacts with the LPS of E.coli.The peptide folds into a kinked helix upon interaction with LPS. The peptide shows very low hemolytic activity.