Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids

Abstract

This report describes a method suitable for determining the depth of a wide variety of fluorescent molecules embedded in membranes. The method involves determination of the parallax in the apparent location of fluorophores detected when quenching by phospholipids spin-labeled at two different depths is compared. By use of straightforward algebraic expressions, the method allows calculation of depth in angstroms. Furthermore, the analysis can be extended to quenching by energy-transfer acceptors or brominated probes under appropriate conditions. Application of the method to quenching of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipids by spin-labeled lipids located at three different depths is demonstrated in model membranes. It is shown that the calculated depths of the NBD groups are self-consistent to the extent that they are the same no matter which two spin-labels have been used in a particular experiment. In addition, the calculated depth is independent of spin-label concentration in the membrane within +/- 1 A, ruling out major effects due to spin-label perturbation. The quenching experiments show that the location of the NBD group in head-group-labeled phosphatidylethanolamine is at the polar/hydrocarbon interface and that of an NBD label on the "tail" of cholesterol is deeply buried, as expected. Unexpectedly, NBD labels placed at the end of fatty acyl chains of phosphatidylcholines are also near the polar/hydrocarbon interface. Presumably, the polarity of the NBD group results in "looping" back to the surface of the NBD groups attached to flexible acyl chains.

Fluorophore environments in membrane-bound probes: A red edge excitation shift study

“…This figure shows dependence of the emission spectrum on the excitation wavelength for NBD-PE. As shown in the figure, upon excitation at 465 nm, the maximum of fluorescence emission2 occurs around 530 nm, as has been observed previously (Chattopadhyay & London, 1988). Changing the excitation wavelength to 500 nm and then to 510 nm causes a progressive shift in the wavelength of maximum emission from 530 to 537 nm.…”

“…Furthermore, these studies show that the average environment of NBD-PE, as monito by the absolute position remains unaffected by these changes. These results are not particularly surprising in view of the fact that changes in membrane organization brought about by phase transition (or temperature) are largely restricted to the fatty acyl region of the membrane (Lentz et al, 1976) and are not sensed by the NBD moiety, which is linked to the headgroup in the case of NBD-PE and is located at the membrape interface (Chattopadhyay & London, 1987Paejano & Martin, 1988;Chattopadhyay, 1990;Mitra & Hammes, 1990;Wolf et al, 1992). It has been previously reported that the rates of solvent relaxation for probes that are not qeeply buried in the hydrocarbon interior of the membrane are more or less independent of temperature (Lakowicz et al, 1983a;takowicz & Keating-Nakamoto, 1984).…”

Behaviour of NBD-head group labelled phosphatidylethanolamines in POPC bilayers: a molecular dynamics study

“…76,80 For NBD-diC n PE, in particular, fluorescence quenching studies have indicated conflicting transverse locations. Using the parallax method, Chattopadhyay and London reported a transverse distance z = 1.42 nm between the fluorophore of NBDdiC 16 PE and the centre of DOPC membranes, 9 which was later revised to the considerably more external values z = 1.89 nm 10 and 2.03 nm, 81 obtained with different combinations of spinlabelled quenchers. These large differences cast important doubts about the reliability of parallax quenching depth determinations, which assume drastic approximations, such as purely static quenching and precise transverse location of two reference quenchers (which must be accurately known), and uses large membrane concentrations of the latter (typically 10 mol%).…”

Interaction of Serotonin1AReceptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

“…6). The NBD group in NBD-PE is localized ∼19Å from the center of the bilayer and reports the head group region of the membrane (Abrams and London, 1993;Chattopadhyay and London, 1987). TMA-DPH is a derivative of DPH with a cationic moiety attached to the para position of one of the phenyl rings (Prendergast et al, 1981).…”

“…TMA-DPH is a derivative of DPH with a cationic moiety attached to the para position of one of the phenyl rings (Prendergast et al, 1981). While DPH is known to partition into the hydrophobic core of the membrane, the amphipathic TMA-DPH is oriented in the membrane bilayer with its positive charge localized at the lipid-water interface (Sojcic et al, (Abrams and London, 1993;Chattopadhyay and London, 1987). The location of the DPH group in TMA-DPH and the average position of DPH in the membrane is shown according to Kaiser and London (1998).…”