Quantification of Protein-Protein Interactions within Membranes by Fluorescence Correlation Spectroscopy

Abstract

The characterization of interactions between membrane proteins as they take place within the lipid bilayer poses a technical challenge, which is currently very difficult and, in many cases, impossible to overcome. The recent development of a method based in the combination two-color fluorescence cross-correlation spectroscopy with scanning of the focal volume allows the detection and quantification of interactions between biomolecules inserted in biological membranes. This powerful strategy has allowed the quantitative analysis of diverse systems, such as the association between proteins of the Bcl-2 family involved in apoptosis regulation or the binding between a growth factor and its receptor during signaling. Here, we review the last developments to quantify protein/protein interactions in lipid membranes and focus on the use of fluorescence-correlation-spectroscopy approaches for that purpose.

GPR37 Protein Trafficking to the Plasma Membrane Regulated by Prosaposin and GM1 Gangliosides Promotes Cell Viability

“…Analysis of Cross-correlation Curves and Interpretation of Cross-correlation Decay Times-Dual-color FCCS relies on the usage of spectrally distinct fluorescent markers and the co-variance of their signals to establish complex formation (24,33,34). The cross-correlation curves showed two characteristic decay times and were fitted using a two-component diffusion model without triplet contribution.…”

“…GPR37 tGFP interactions with PS-TX14(A) TAMRA at the plasma membrane were further characterized using FCS and dual-color FCCS. FCCS can detect complex formation in living cells in a manner that is independent of distance between the fluorophores by measuring the co-variance of fluorescence intensity fluctuations of the dually labeled complexes (24,33,34).…”

Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

“…Sophisticated protein–protein interaction networks have been assembled over the past decades by combining genetic and biochemical assays, including yeast two-hybrid screens, coimmunoprecipitation, and pull-down experiments (Barrios-Rodiles et al, 2005; Suter et al, 2008; Vermeulen et al, 2008), yet robust methods for validation and quantification of interactions involving membrane proteins under physiological conditions are still missing. Current techniques for quantitative protein interaction analysis in live cells, such as Förster resonance energy transfer (Day and Davidson, 2012; Padilla-Parra and Tramier, 2012; Sun et al, 2012) or fluorescence cross-correlation spectroscopy (Kim et al, 2007; Bleicken et al, 2011; Ries and Schwille, 2012) are highly demanding and often fail in case of low affinity, transient interactions, or large multiprotein complexes in the context of membranes. For these reasons, quantitative analysis of interactions within transmembrane signaling complexes remains particularly challenging.…”

Plasmonic Nanosensors for Simultaneous Quantification of Multiple Protein–Protein Binding Affinities

“…The dynamics in living organisms is governed by a complex network of interacting macromolecules. − For example, in a process like cell division a dozen of proteins interacts in a subtle and interconnected way of subprocesses to finally define a division place or to generate the constriction force in the cell wall. − To fully understand, model, and potentially influence such a process, it is important to quantify binding affinities between all possible partners, preferentially without introducing fluorescent labels. Popular techniques to do so are surface plasmon resonance (SPR) biosensors, quartz crystal microbalance, fluorescence correlation spectroscopy, isothermal titration calorimetry, or analytical ultracentrifugation . However, most of the label-free techniques used to study binding affinities require the analysis of one pair of binding partners at a time, making the quantification of a complex interaction network a laborious and slow effort.…”

“…4−6 To fully understand, model, and potentially influence such a process, it is important to quantify binding affinities between all possible partners, preferentially without introducing fluorescent labels. Popular techniques to do so are surface plasmon resonance (SPR) biosensors, 7 quartz crystal microbalance, 8 fluorescence correlation spectroscopy, 9 isothermal titration calorimetry, 10 or analytical ultracentrifugation. 11 However, most of the label-free techniques used to study binding affinities require the analysis of one pair of binding partners at a time, making the quantification of a complex interaction network a laborious and slow effort.…”