Direct Quantification of Serum Protein Interactions with PEGylated Micelle Nanocarriers.

A large repertoire of nanocarrier (NC) technologies exists, each with highly specified advantages in terms of targetability, stability, and immunological inertness. The characterization of such NC properties within physiological conditions is essential for the development of optimized drug delivery systems. One method that is well established for reducing premature elimination by avoiding protein adsorption on NCs is surface functionalization with poly(ethylene glycol) (PEG), aptly called PEGylation.

Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells.

Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy.

Fluorescence cross-correlation spectroscopy (FCCS) is an advanced fluorescence technique that can quantify protein-protein interactions in vivo. Due to the dynamic, heterogeneous nature of the membrane, special considerations must be made to interpret FCCS data accurately. In this study, we describe a method to quantify the oligomerization of membrane proteins tagged with two commonly used fluorescent probes, mCherry (mCH) and enhanced green (eGFP) fluorescent proteins.

A G Protein-Coupled Receptor Dimerization Interface in Human Cone Opsins.

G protein-coupled receptors (GPCRs) detect a wide variety of physical and chemical signals and transmit that information across the cellular plasma membrane. Dimerization is a proposed modulator of GPCR signaling, but the structure and stability of class A GPCR dimerization have been difficult to establish. Here we investigated the dimerization affinity and binding interface of human cone opsins, which initiate and sustain daytime color vision.

Molecular basis for multimerization in the activation of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR.

Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface.

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment.

Tuning the mobility coupling of quaternized polyvinylpyridine and anionic phospholipids in supported lipid bilayers.

Binding of biomacromolecules to anionic lipids in the plasma membrane is a common motif in many cell signaling pathways. Previous work has shown that macromolecules with cationic sequences can form nanodomains with sequestered anionic lipids, which alters the lateral distribution and mobility of the membrane lipids. Such sequestration is believed to result from the formation of a lipid-macromolecule complex.