Investigation into the membrane interactive properties of the escherichia coli low molecular weight penicillin-binding proteins

Various results have suggested that in Escherichia coli murein assembly may involve a protein complex(es) which could include low molecular mass penicillin-binding prpteins (PBPs). These proteins include PBP4, PBP5 and PBP6 which are penicillin sensitive enzymes associated with the periplasmic face of the inter membrane. The levels of these associations have been linked to enzymic activity and elucidation of the mechanism(s) involved in these associations may help identify and understand the regulation of this putative protein complex. It is currently accepted that the membrane associations of PBP5 and PBPÔ involve C-terminal amphiphilic cz-helices and such helices are ubiquitously employed in the lipid associations of membrane interactive protein molecules. Whether such helical structure features in the membrane associations of PBP4 or indeed if this protein is membrane bound or soluble, are, as yet, open questions. The focus of this research has been to investigate the lipid and membrane interactions of PBP4, PBP5 and PBP6 and in particular, to investigate the role played by these interactions of the C-terminal region of these proteins. Haemolytic analysis has shown that peptide homologues of the PBP5 and PBP6 C-terminal regions, P5 and P6, are active at the membrane interface and CD analysis has shown that these peptides possess a capacity for a-helix formation. CD and pressure - area isotherm analysis of monolayers formed from PS and P6 have shown that these peptides are able to adopt a-helical structure at an air - water interface. Monolayer studies have shown that P5 and P6 are able to interact with lipids and that these interactions are characterised by minor requirements for anionic lipid and the involvement of predominantly hydrophobic forces which are enhanced by low pH. Similar characteristics were revealed when perturbant washes and Western blotting were used to investigate the interactions of PBP5 with membranes derived from a mutant E. coli strain, HDL 11, in which the level of anionic lipid can be controlled. Overall, these results strongly support the hypothesis that C-terminal amphiphilic a-helices feature in PBP5 and PBP6 membrane anchoring. Molecular area determinations have implied that a peptide homologue of the PBP4 C-terminal region, P4 is able to adopt a-helical structure and this was confirmed by CD analysis. P4 showed no haemolytic activity but the peptide was found to interact generally with lipid monolayers. These monolayer interactions were characterised by a requirement for anionic lipid and involved predominantly electrostatic forces, which were enhanced by low pH. Similar characteristics but with no detectable requirement for anionic lipid were revealed when perturbant washes and chemiluminesence were used to investigate the interactions of PBP4 with membranes of the overproducing strain HB 10 I/pBK4 and those of HDL1 1. It is suggested that the PBP4 C-terminal region may play a role in PBP4 - membrane anchoring. Using chemiluminesence, no soluble form of PBP4 could be detected in the wild type E. coli, MRE600, suggesting that in wild type strains, PBP4 is exclusively membrane bound. It is suggested that PBP4 - membrane anchoring occurs at a specific binding site and overall, may differ fundamentally from that of PBP5 and PBP6