Frederick Harrisa, Andre Wieseb, Klaus Brandenbergb, David. Phoenixa* and Ulrich Seydelb. aDepartment of Applied Biology, University of Central Lancashire,.
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Biochemical Society Transactions (1998) 26 An investigation into the lipid interactions of peptides correaponding to the C-terminal anchoring domains of Esckenkhb cdi penicillin-binding proteins 4 and 5.
Frederick Harrisa, Andre Wieseb, Klaus Brandenbergb, David Phoenixa* and Ulrich Seydelb. aDepartment of Applied Biology, University of Central Lancashire, Preston, PRI 2HE and &vision of Biophysics, Research Centre Borstel, Parkallee 10, D-23845 Borstel, Germany. The Escherichia coli penicillin-binding proteins (PBPs), PBP4 and PBPS are enzymes involved in murein biosynthesis and are anchored to the periplasmic hce of the inner membrane [I]. It has been predicted that for PBPS this anchoring could involve an amphiphilic C-terminal a-helii but that for PBP4, anchoring may involve the C-terminal region in either an amphiphilic a-helical or an amphiphilic p-sheet conformation [2] and here, we have sought to test these predictions. We have utilised Fourier transform infrared spectroscopy to study the secondary structures of peptide homologues of the PBP4 and PBPS C-terminal regions, P4 and P5 (Table 1). We have investigated the ability of P4 and PS to intercalate with liposomes double labelled with NBD-PE (N-(-7-nitr0-2,1,3benzoxadiazol-4-yl)-phosphatidylethanolamine) and Rh-PE (N(rhodamine B sulphonyl>phosphatidylethanolamine, using the resonance energy transfer technique and the ability of P4 and PS to interact with black lipid membranes (BLM) by following changes in conductivity. Infra red spectroscopy indicated that for the pure peptide and in the presence of vesicles formed from either dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylglycerol (DMPG), the secondary structure of PS is predominantly a-helical. Intensity increases observed with double labelled liposomes formed from either naturally Occuring phosphatidylserine (PS) or phosphatidylcholine(PC) seemed to indicate that at neutral pH, PS was able to intercalate with both lipids. The interaction of PS with BLM formed fiom dioleoyl phosphatidylglycerol (DOPG) showed stepwise conductivity changes which are consistent with the insertion of PS into the membrane. Furthermore, at low concentrations PS was found to cause an asymmetry in the intrinsic membrane voltage of BLM formed from diphytanoyl phosphatidylcholine (DPhyPC), suggesting an attachment of PS to the membrane, However, at higher concentrations PS caused the immediate rupture of DPhyPC membranes. Overall these tindings show that P5 is able to interact with both anionic and zwitterionic lipid layers and suggest that these interactions could involve ahelical structure in the peptide. Furthermore, these &dings suggest that depending upon conditions, these interactions may be of a specific nature and could involve an ordered insertion of P5 into the lipid layer Infrared spectroscopy revealed that for the pure peptide and in the presence of vesicles formed fiom either DMPC or DMPG, pstrands and p-turns made the major contribution to the secondary structure of P4. Intensity increases observed with double labelled PS liposomesseemed to indicate that k was able to intercalate Table 1. The ~rimarvstructures of -tides
P4 and PS
The peptides are homobgous to the C-termini of proteins PBP4 [3] and PBPS [4] respectively.
PBPS
G$FGKIID-G-COOH
with the lipid and that levels of intercalation became progressively greater with increasing pH in the range of pH 4 to pH 8. However, no intercalation of P4 could be detected with labelled PC liposomes. P4 was also found to cause the immediate rupture of BLM formed from either PS or DPhyPC, with no asymmetry of intrinsic membrane potential. Overall, these findings show that depending upon conditions, P4 is able to interact with both anionic and zwitterionic lipid layers. However, the interactions of P4 seem to be of a less specific nature than those of P5 and may involve p type structures in the peptide rather than the a-helical structure observed in PS. In general, these results appear to support theoretical predictions made for the PBP4 and PBPS C-terminal regions and support the view that the PBP4 - membrane anchoring mechanism may differ to that of PBPS. References [l] Pewsey, A. R., Phoenix, D. A. and Roberts, M. G. (1996) Protein Peptide Letts. 3, 185-192. [2] Phoenix, D. A. and Harris, F. (1995) Biochem. SOC.Trans 23, 976-980 [3] Broome-Smith, J. K.,Ionnidis; I., Edelman, A. and Spratt, B. G. (1988), Nucleic Acid Research 16, 1617 [4] Mottl, H., Tepstra, P. and Keck, W., (1991) FEMS Microbiol. Lett. 78.213-220
