Quenching-enhanced fluorescence titration protocol for accurate determination of free energy of membrane binding

Abstract

Fluorescence spectroscopy is a convenient tool to examine peptide-membrane interactions at equilibrium (1) owing to the change in emission properties of many fluorophores (including tryptophan) during transfer from an aqueous environment into a lipid bilayer. In some cases (e.g. mechanosensitive channel blocker GsMTx4 described here), however, binding-associated emission changes are too small for reliable determination of the free energy of partitioning, ΔG. To enhance the spectroscopic response to binding we implemented the titration with lipid vesicles in the presence of aqueous ionic quencher iodide, which preferentially quenchers fluorescence of the free peptide in solution. We have verified the accuracy of this new titration protocol using the well-studied peptide melittin.

Figure 1

Fluorescence emission spectra of 1 μM GsMTx4 in buffer (dashed lines) and in the presence of 75POPC:25POPG large unilamellar vesicles (LUV) containing 0.8 mM lipid (solid lines). Intensity changes, associated with membrane partitioning, differ substantially depending on the absence (A) or presence (B) of ionic quencher I in the buffer. Because of the preferential quenching of fluorescence of aqueous peptide, the presence of 50 mM KI enhances the signal associated with membrane partitioning.

Figure 2

Testing (upper panel) and applying (lower panel) quenching-enhanced titration protocol for determining the free energy of membrane partitioning, ΔG. The well-studied peptide melittin was titrated with POPC LUV in the presence of 50 mM of either KCl (upper panel, open squares) or KI (upper panel, solid squares). Fitting of the titration data with Eqs. 1–2 (curves) demonstrates invariability of ΔG in the two samples, while the fluorescence increase accompanying membrane partitioning (I_max)is markedly increased in the presence of KI. This enhanced signal sensitivity, achieved in the presence of 50 mM KI, allows accurate determination of the ΔG of partitioning of GsMTx4 into membranes of various lipid compositions, such as zwitterionic POPC vesicles (lower panel, circles) and anionic 25POPC:75POPG vesicles (lower panel, triangles).

Figure 2

Testing (upper panel) and applying (lower panel) quenching-enhanced titration protocol for determining the free energy of membrane partitioning, ΔG. The well-studied peptide melittin was titrated with POPC LUV in the presence of 50 mM of either KCl (upper panel, open squares) or KI (upper panel, solid squares). Fitting of the titration data with Eqs. 1–2 (curves) demonstrates invariability of ΔG in the two samples, while the fluorescence increase accompanying membrane partitioning (I_max)is markedly increased in the presence of KI. This enhanced signal sensitivity, achieved in the presence of 50 mM KI, allows accurate determination of the ΔG of partitioning of GsMTx4 into membranes of various lipid compositions, such as zwitterionic POPC vesicles (lower panel, circles) and anionic 25POPC:75POPG vesicles (lower panel, triangles).