Coexistence of domains with distinct order and polarity in fluid bacterial membranes.
In this study we sought the detection and characterization of bacterial membrane domains. Fluorescence generalized polarization (GP) spectra of laurdan-labeled Escherichia coli and temperature dependencies of both laurdan's GP and fluorescence anisotropy of 1,3-diphenyl-1,3,5-hexatriene (DPH) (rDPH) affirmed that at physiological temperatures, the E. coli membrane is in a liquid-crystalline phase. However, the strong excitation wavelength dependence of rlaurdan at 37 degrees C reflects membrane heterogeneity. Time-resolved fluorescence emission spectra, which display distinct biphasic redshift kinetics, verified the coexistence of two subpopulations of laurdan. In the initial phase, <50 ps, the redshift in the spectral mass center is much faster for laurdan excited at the blue edge (350 nm), whereas at longer time intervals, similar kinetics is observed upon excitation at either blue or red edge (400 nm). Excitation in the blue region selects laurdan molecules presumably located in a lipid domain in which fast intramolecular relaxation and low anisotropy characterize laurdan's emission. In the proteo-lipid domain, laurdan motion and conformation are restricted as exhibited by a slower relaxation rate, higher anisotropy and a lower GP value. Triple-Gaussian decomposition of laurdan emission spectra showed a sharp phase transition in the temperature dependence of individual components when excited in the blue but not in the red region. At least two kinds of domains of distinct polarity and order are suggested to coexist in the liquid-crystalline bacterial membrane: a lipid-enriched and a proteolipid domain. In bacteria with chloramphenicol (Cam)-inhibited protein synthesis, laurdan showed reduced polarity and restoration of an isoemissive point in the temperature-dependent spectra. These results suggest a decrease in membrane heterogeneity caused by Cam-induced domain dissipation.
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Sharon
S
Vanounou
Dina
D
Pines
Ehud
E
Pines
Abraham H
AH
Parola
Itzhak
I
Fishov
D015081:2-Naphthylamine / Q000031:analogs & derivatives*; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects; D002701:Chloramphenicol / Q000494:pharmacology; D004161:Diphenylhexatriene; D004926:Escherichia coli / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007848:Laurates; D008563:Membrane Lipids / Q000737:chemistry; D018462:Photobiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013816:Thermodynamics
D005456:Fluorescent Dyes; D007848:Laurates; D008563:Membrane Lipids; D011500:Protein Synthesis Inhibitors; D004161:Diphenylhexatriene; D002701:Chloramphenicol; D015081:2-Naphthylamine; C065580:laurdan