Using a Novel Dual Fluorescence Quenching Assay for Measurement of Tryptophan Depth within Lipid Bilayers To Determine Hydrophobic α-Helix Locations within Membranes

Abstract

A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polypeptides is introduced. Quenching of Trp by acrylamide and 10-doxylnonadecane (10-DN) was used to measure Trp depth. Transmembrane helices with Trp residues at varying positions (and thus locating at different depths in lipid bilayers) were used to calibrate the method. It was found that acrylamide quenches Trp close to the bilayer surface more strongly than it quenches deeply buried Trp, while 10-DN quenches Trp close to the center of the bilayer more strongly than Trp close to the surface. The ratio of acrylamide quenching to that of 10-DN was found to be nearly linearly dependent on the depth of Trp in a membrane. It was also found that it was possible to detect coexisting shallowly and deeply inserted populations of Trp-containing polypeptides using these quenchers. In the presence of such mixed populations, acrylamide induced large blue shifts in fluorescence emission lambda(max) whereas 10-DN induced large red shifts. In a more homogeneous population quencher-induced shifts were found to be minimal. Dual quencher analysis can be used to distinguish hydrophobic helices with a transmembrane orientation from those located close to the bilayer surface and, when applied to a number of different peptides, revealed novel aspects of hydrophobic helix behavior.

Mechanism for HIV-1 Tat Insertion into the Endosome Membrane

“…To prepare brominated vesicles, half of the PC was replaced by 1,2-(9,10-dibromo)stearoyl phosphatidylcholine (24). When indicated the 10-DN quencher was added at 10 mol % before drying the lipids (36). Experiments were performed using 2 M Tat in citrate buffer, pH 7.0, unless otherwise stated.…”

“…Tat is able to insert into model membranes upon acidification, and if Trp-11 is significantly involved in this insertion process, it should directly contact lipids and therefore be accessible to membrane-embedded quenchers such as dibromo-PC (24) or 10-doxylnonadecane (10-DN) (36). In the presence of control lipid vesicles, some Tat Trp quenching took place during acidification, but this quenching was relatively weak and plateaued below pH 5.7 (Fig.…”

“…We then assessed whether Tat basic domain mutants could insert into model membranes upon acidification. We used lipid vesicles and membrane-embedded quenchers, distearoylbromo-PC, which bears bromide atoms in positions 9 and 10, and 10-DN, which is a highly hydrophobic molecule containing a nitroxide and that is thought to distribute homogeneously within the membrane core (36). The resulting quenching curves (Fig.…”

Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

“…1B) was used to study the ability of the hydrophobic sequence to span lipid bilayers. Fluorescence methods we developed previously were used to define the topography of the bilayer-inserted sequences (43)(44)(45)(46)(47)49 To measure Trp depth directly, a dual fluorescence quenching method was used (47). In this method the ratio of quenching of Trp by acrylamide, which resides in the aqueous solution, to quenching by the membrane-inserted quencher 10-doxylnonadecane is measured.…”

“…In this method the ratio of quenching of Trp by acrylamide, which resides in the aqueous solution, to quenching by the membrane-inserted quencher 10-doxylnonadecane is measured. This quenching ratio (Q-ratio) responds nearly linearly to Trp depth in the bilayer, such that a low quenching ratio (<0.15) indicates a deeply located Trp near the center of the bilayer, while a high Q-ratio (>1) is indicative of a Trp near the bilayer surface (45,47). Figure 3 and Table 3 show values for anchor peptide-1 Trp λmax and how it is affected by bilayer width (varied by using lipids with different length acyl chains).…”