Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy

Abstract

Fluorescence Correlation Spectroscopy (FCS) has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners however comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a binding model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants D K . The total protein concentration, but not the fraction of labeled protein needs to be known. In this work, we apply our dcFCCS analysis scheme to Sar1p, a protein of the COPII complex, which binds 'majorminor-mix' liposomes. A Langmuir isotherm model yieldsas the single site dissociation constant. The dual-color FCCS framework presented here is highly versatile for biophysical analysis of binding interactions. It may be applied to many types of fluorescently labeled ligands and small diffusing particles, including nanodiscs and liposomes containing membrane protein receptors.

α-Helical peptidic scaffolds to target α-synuclein pathogenic species with high affinity and selectivity

“…We next performed a titration experiment for analyzing the binding of the peptide to the three αS aggregated species. For this, we developed a model-independent saturation binding curve, based on the theoretical framework previously developed by Kruger and coworkers 31 . This analysis allowed us to quantify the number of peptide molecules bound to each αS species ( N p ) as a function of the concentration of unbound peptide ( C p,Free ) ( Figure 2e ).…”

Measuring Protein Insertion Areas in Lipid Monolayers by Fluorescence Correlation Spectroscopy

“…The GTPase Sar1p from Saccharomyces cerevisiae was expressed and purified as previously described (24,27). The protein variant Sar1pS147C/C171S was prepared in the same way, but additionally labeled with Alexa Fluor 488 maleimide (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA).…”

“…This position lies on the membrane-distal surface and thus does not interfere with membrane binding. Both labeled and unlabeled Sar1p proteins were functional in a membrane binding assay (24) and with respect to GTPase enzyme activity. Protein concentrations were determined by UV absorption, using linear unmixing of the protein spectrum and the nucleotide spectrum, because Sar1p carries a GDP molecule in its active center.…”

“…Using fluorescently labeled proteins and membrane constituents, Fluorescence Cross-Correlation Spectroscopy (FCCS) is used to quantify binding affinities of proteins to bilayers in the form of small vesicles. FCCS yielded a low micromolar Kd for Sar1p binding to majorminor-mix lipids (24). Here, we complement this FCCS application by quantifying the insertion areas upon binding in monolayers using the same protein and the same lipid composition.…”

A facile, sensitive and quantitative membrane‐binding assay for proteins

“…Understanding their functions requires analyzing their membrane binding properties on compositionally well‐defined membrane mimetic systems, for which there exists a variety of assays . These range in sophistication from relatively simple lipid dot‐blots, pull‐downs with lipids immobilized on beads, pull‐down of liposomes to immobilized proteins and liposome co‐sedimentation assays to finer and technically‐elaborate single liposome‐based detection methods . Despite their utility, these methods typically represent a trade‐off between fidelity of model systems to mimic native membranes, sensitivity and throughput.…”