How Different Lipidic Mimetic Systems of E. COLI Membranes Affect Membrane Protein Reconstitution: A Steady-State Fluorescence Anisotropy Study

Abstract

Outer membrane proteins (Omp) in bacteria, porins, have an important role on antibiotic translocation trough bacterial membranes. OmpF is one of the most important E. coli porins in the permeation of beta-lactam and quinolone antibiotics. Moreover, these antibiotics might penetrate the bacterial outer membrane through porins or through the lipid/protein interface. We focused on the characterization of E. coli membranes, once this knowledge is crucial for studies of antibiotic translocation and useful to understand and counteract bacterial antibiotic resistance, one of the biggest concerns of nowadays. Steady-state fluorescence anisotropy is a good technique to detect changes in lipid environments through the use of fluorescent probes. We used two fluorescent probes, DPH and TMA-DPH (which provide information from the core and from the interface regions of the bilayer, respectively), and determined the temperature dependence of these two anisotropy probes, incubated or incorporated into POPE/POPG (0.75:0.25), POPE/POPG/Cardiolipin (0.67:0.23:0.10) and E. coli total lipid extract liposomes (LUVs) and OmpF proteoliposomes. POPE/POPG and POPE/POPG/Cardiolipin mixtures are conventionally used to mimic the E. coli membranes, while the E. coli total lipid extract is a natural system. In this study we determine the phase transition temperatures characteristics of each membrane mimetic system and still prove the correct insertion of the protein in the membrane. When comparing the transition temperatures obtained for each mimetic system, our study suggest that the ternary lipid mixture is a better system to mimic the E. coli membranes than the binary system.