Fluorescence quenching and electron spin resonance study of percolation in a two-phase lipid bilayer containing bacteriorhodopsin

Abstract

The effect of bacteriorhodopsin (BR) on the percolation properties of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine bilayers was examined by studying the quenching of a lipid-bound fluorophore by a lipid-bound quencher, and by spin-spin interactions of a nitroxide-labeled lipid using electron spin resonance (ESR). At the low concentrations of BR used, differential scanning calorimetry showed that although the transition enthalpy was reduced in a concentration-dependent manner by incorporation of BR, the solidus and fluidus phase boundaries and overall shape of the heat capacity profiles were essentially unchanged. However, fluorescence quenching and spin-label ESR data showed that the domain topology, as reflected in the percolation properties, is strongly affected by the protein. In contrast to our previous fluorescence data for the pure lipid mixtures, quenching in the coexistence region is independent of the fluid phase fraction when BR is present. In addition, the percolation threshold estimated by spin-label ESR is shifted in the presence of BR to a higher gel phase fraction at a given lipid composition. Both the fluorescence quenching and spin-label ESR data, together with the results of earlier simulations, strongly suggest that the fluid phase domains are substantially larger and/or less ramified in the presence of BR than in its absence. We have previously reported a similar effect of a transmembrane peptide, pOmpA (Escherichia coli outer membrane protein A signal peptide), on fluid domain connectivity in binary phosphatidylcholine mixtures.

Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers

“…Tryptophan fluorescence quenching by brominated phospholipids (Alvis et al, 2003;Bolen and Holloway, 1990;Everett et al, 1986;Ladokhin, 1999aLadokhin, , 1999bLadokhin and Holloway, 1995;Markello et al, 1985;Williamson et al, 2002) or by lipid spin-labels (Abrams andLondon, 1992, 1993;Cruz et al, 1998;Fastenberg et al, 2003;Piknova et al, 1997;Prieto et al, 1994) has been very valuable to determine characteristic elements of the transmembrane topology and lipid-protein interactions of integral membrane proteins. To further characterize the folding process of OmpA, we combined this method with the study of the folding kinetics of OmpA into bilayers (SUVs) of diC 18:1 PC (Kleinschmidt et al, 1999a;Kleinschmidt and Tamm, 1999).…”

Folding and Stability of Monomeric β‐Barrel Membrane Proteins

“…[75][76][77][78][79][80][81][82]) or by lipid spin-labels (see, e.g. [83][84][85][86][87][88]) traditionally has been very valuable to determine characteristic elements of the transmembrane topology and lipidprotein interactions of integral membrane proteins. To further characterize the folding process of OmpA, we combined this method with the study of the folding kinetics of OmpA into bilayers (SUVs) of diC 18 : 1 PC [89,90].…”

Assembly of Integral Membrane Proteins from the Periplasm into the Outer Membrane

“…Tryptophan fluorescence quenching by brominated phospholipids (see, e.g., Alvis et al, 2003;Bolen and Holloway, 1990;Everett et al, 1986;Ladokhin and Holloway, 1995;Ladokhin, 1999a, b;Markello et al, 1985;Williamson et al, 2002) or by lipid spin labels (see, e.g., London, 1992, 1993;Cruz et al, 1998;Fastenberg et al, 2003;Piknova et al, 1997;Prieto et al, 1994) traditionally has been very valuable to determine characteristic elements of the transmembrane topology and lipid-protein interactions of TMPs.…”