Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop antibacterial agents with novel modes of action. A promising alternative to antibiotics are the naturally occurring endolysin enzymes from bacteriophage. Endolysins cause bacterial lysis by degrading the bacterial peptidoglycan cell wall. Exogenous application of endolysins results in rapid and specific elimination of Gram-positive bacteria making them an excellent alternative and / or adjunct to traditional antibiotics. The streptococcal C1 phage lysin, PlyC, is the most potent endolysin described to date and can rapidly lyse Group A, C and E Streptococci [1]. We have previously determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins, constituting a multimeric cell-wall docking assembly bound to two catalytic domains [2]. However, the crystal structure appeared to be auto- inhibited and raised important questions as to the mechanism underlying the extreme potency of PlyC [3]. Here, we present our latest research incorporating circular dichroism spectroscopy and hydrogen-to-deuterium exchange detected by mass spectrometry to investigate the stability and inter-domain communication in PlyC. Further, we have initiated a directed evolution study to engineer PlyC to target other groups of Streptococci. Together, these results will provide valuable insight into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency and how this lytic activity can be used to kill different bacterial strains.