Slow rotational mobilities of antibodies and lipids associated with substrate-supported phospholipid monolayers as measured by polarized fluorescence photobleaching recovery

Abstract

Polarized fluorescence photobleaching recovery has been used to monitor slow rotational motions of a fluorescently-labeled anti-dinitrophenyl mouse IgGl monoclonal antibody (ANO2) specifically bound to substrate-supported monolayers composed of a mixture of distearoylphosphatidylcholine (DSPC) and dinitrophenyldioleoylphosphatidylethanolamine (DNP-DOPE). ANO2 antibodies were labeled with a new bifunctional carbocyanine fluorophore that has two amino-reactive groups; steady-state fluorescence anisotropy data confirmed the expected result that the ANO2-conjugated bifunctional probe had less independent flexibility than ANO2-conjugated unifunctional fluorescence labels. Rotational mobilities were also measured for the fluorescent lipid 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine (dil) in DSPC and in mixed DSPC/DNP-DOPE monolayers in the presence and absence of unlabeled ANO2 antibodies. The apparent rotational correlation time and fractional mobility of ANO2 on supported monolayers were approximately 70 and approximately 0.3 s, respectively. These measured parameters of rotational mobility did not depend on the ANO2 surface density or on kinetic factors, but addition of unlabeled polyclonal anti-(mouse IgG) antibodies significantly decreased the apparent mobile fraction. The measured fluorescence recovery curves for dil were consistent with two fluorophore populations with rotational correlation times of approximately 4 and approximately 100 s and a population of immobile fluorescent lipid. No difference in fluorescence recovery and decay curves was measured for dil in DSPC monolayers, DSPC/DNP-DOPE monolayers, and DSPC/DNP-DOPE monolayers treated with unlabeled ANO2 antibodies.

Combinatorial microscopy for the study of protein–membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM

“…2A and B). This observation was in agreement with the fact that this probe is known to embed in lipid bilayers with the linear conjugated bridge of the molecule (i.e., the probe chromophore) aligned horizontally with respect to the membrane plane such that the alkyl chains connected to the bridge can insert into the hydrophobic membrane core (Axelrod, 1979;Sund et al, 1999;Timbs and Thompson, 1990). This orientation was supported by recent molecular dynamics simulations of a related fluorescent probe DiI-C 18 in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 16:0) lipid bilayers (Gullapalli et al, 2008).…”

“…The technique of polarized total internal reflection fluorescence microscopy (pTIRFM) is able to infer the time-and ensemble-averaged orientational order related to a collection of fluorescent probe molecules located within an interfacial system such as a substrate-supported bilayer through a quantity known as the orientational order parameter, hP 2 i (Axelrod, 1989(Axelrod, , 2001Burghardt, 1984;Oreopoulos and Yip, 2009;Sund et al, 1999;Thompson et al, 1984;Timbs and Thompson, 1990). This order parameter is similar to those encountered in other orientation-sensitive spectroscopies and has an analogous interpretation Bolterauer and Heller, 1996;Douliez et al, 1998;Lafrance et al, 1995;Lopes and Castanho, 2005;Schafer et al, 1998;Vermeer et al, 2007).…”

Fluorescence Spectroscopy in Biology

“…A very elegant variant is the combination of FRAP with the total internal reflection fluorescence (TIRF) technique (total internal reflection fluorescence recovery after photobleaching; TIR-FRAP [28]) which is covered in greater depth in the contribution of N. Thompson (Chap. Using planar phospholipid bilayers and fluorescently labelled proteins, this method allows the determination of adsorption/desorption rate constants and surface diffusion constants [28,131,132]. Here, a laser beam totally internally reflects at a solid/liquid interface, creating an evanescent field which penetrates only a fraction of the laser beam's wavelength into the liquid domain.…”

“…In the simplest form of this method, the linear absorption dichroism is measured by examining the dependence of the evanescently excited fluorescence on the polarization of the evanescent field. P-TIRFM has been experimentally applied to a variety of fluorescent lipids in supported planar membranes [91,129,[131][132][133] as well as cytochrome c at dif-ferent surfaces [134][135][136][137][138] and plasminogen at modified surfaces [139]. 6.1).…”

Lateral mobility of phospholipid molecules in thin liquid films

“…Schlessinger et al 1976Schlessinger et al , 1977Ishida et al 1993;Greenberg and Axelrod 1993;Eldridge et al 1980) and in model membrane systems, for instance in phospholipid monolayers (e.g. Beck and Peters 1985;Tombs and Thompson 1990), bilayer lipid membranes (BLMs) (Fahey et al 1977).…”