Abstract
Understanding the interplay between molecules and lipid membranes is fundamental when studying cellular and biotechnological phenomena. Partition between aqueous media and lipid membranes is key to the mechanism of action of many biomolecules and drugs. Quantifying membrane partition, through adequate and robust parameters, is thus essential. Surface Plasmon Resonance (SPR) is a powerful technique for studying 1:1 stoichiometric interactions but has limited application to lipid membrane partition data. We have developed and applied a novel mathematical model for SPR data treatment that enables determination of kinetic and equilibrium partition constants. The method uses two complementary fitting models for association and dissociation sensorgram data. The SPR partition data obtained for the antibody fragment F63, the HIV fusion inhibitor enfuvirtide, and the endogenous drug kyotorphin towards POPC membranes were compared against data from independent techniques. The comprehensive kinetic and partition models were applied to the membrane interaction data of HRC4, a measles virus entry inhibitor peptide, revealing its increased affinity for, and retention in, cholesterol-rich membranes. Overall, our work extends the application of SPR beyond the realm of 1:1 stoichiometric ligand-receptor binding into a new and immense field of applications: the interaction of solutes such as biomolecules and drugs with lipids.
Highlights
Membrane bilayers to a bulk lipid phase
The detected CF fluorescence signal intensity was close to background noise and did not co-localize with Rho-PE, suggesting that the probe was released during Small unilamellar vesicles (SUV) deposition
We have developed a SPR analytical methodology to assess solute partition towards lipid membranes
Summary
Membrane bilayers to a bulk lipid phase. The steady-state model allows Kp determination from sensorgram association phase response data when a maximum steady-state response is achieved. The dissociation model provides dissociation rate constants (koff) from kinetic evaluation of dissociation data Both are applied to conventional sensorgram response data from commercial Biacore L1 sensor chip experiments. The experimental design of the present method follows three distinct steps: First we evaluated the surface coverage of Biacore L1 sensor chips by lipid vesicles and vesicles integrity after deposition. This allows experimental validation of specific model assumptions. The integration of steady-state and dissociation models was applied to the binding and unbinding of the HRC4 dimeric peptide, a novel antiviral fusion inhibitor against measles virus (MV) derived from a heptad repeat domain at the C-terminus of the MV fusion (F) protein[17], designed to interact with lipid membranes (Supplementary Table S1). The influence of membrane-induced aggregation in the dissociation mechanism will be discussed
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