Analysis of transient membrane protein interactions by single-molecule diffusional mobility shift assay

Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.Subject terms: Fluorescence imaging, Super-resolution microscopy, Single-molecule biophysics     

Bright,Highly Water‐Soluble Triazacyclononane Europium Complexes To Detect Ligand Binding with Time‐Resolved FRET Microscopy

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water‐soluble, and negatively charged sulfonic‐ or carboxylic acid derivatives of para‐substituted aryl–alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell‐surface proteins with time‐resolved Förster resonance energy transfer (TR‐FRET) assays in plate‐based format and using TR‐FRET microscopy.     

Bright,Highly Water‐Soluble Triazacyclononane Europium Complexes To Detect Ligand Binding with Time‐Resolved FRET Microscopy

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water‐soluble, and negatively charged sulfonic‐ or carboxylic acid derivatives of para‐substituted aryl–alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell‐surface proteins with time‐resolved Förster resonance energy transfer (TR‐FRET) assays in plate‐based format and using TR‐FRET microscopy.     

Single‐Molecule Methods to Study Membrane Receptor Oligomerization

Membrane receptors control fundamental cellular processes. Binding of a specific ligand to a receptor initiates communication through the membrane and activation of signaling cascades. This activation process often leads to a spatial rearrangement of receptors in the membrane at the molecular level. Single‐molecule techniques contributed significantly to the understanding of receptor organization and rearrangement in membranes. Here, we review four prominent single‐molecule techniques that have been applied to membrane receptors, namely, stepwise photobleaching, Förster resonance energy transfer, sub‐diffraction localization microscopy and co‐tracking. We discuss the requirements, benefits and limitations of each technique, discuss target labeling, present a selection of applications and results and compare the different methodologies.     

19F NMR Spectroscopy as a Probe of Cytoplasmic Viscosity and Weak Protein Interactions in Living Cells

Protein mobility in living cells is vital for cell function. Both cytosolic viscosity and weak protein–protein interactions affect mobility, but examining viscosity and weak interaction effects is challenging. Herein, we demonstrate the use of ¹⁹F NMR spectroscopy to measure cytoplasmic viscosity and to characterize nonspecific protein–protein interactions in living Escherichia coli cells. The origins of resonance broadening in Escherichia coli cells were also investigated. We found that sample inhomogeneity has a negligible effect on resonance broadening, the cytoplasmic viscosity is only about 2–3 times that of water, and ubiquitous transient weak protein–protein interactions in the cytosol play a significant role in governing the detection of proteins by using in‐cell NMR spectroscopy.     

Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads

G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.     

TERS Detection of αVβ3 Integrins in Intact Cell Membranes

Integrins are important membrane receptors that form focal adhesions with the extracellular matrix and are transmembrane signaling proteins. We demonstrate that nanoparticles functionalized with c‐RGDfC ligands bind to intact cell membranes and selectively enhance the amino acid signals of the integrin receptor when coupled with tip‐enhanced Raman scattering (TERS) detection. Controlling the plasmonic interaction between the functionalized nanoparticle and the TERS tip provides a clear Raman signal from α_Vβ₃ integrins in the cell membrane that matches the signal of the purified integrin receptor. Random aggregation of nanoparticles on the cell does not provide the same spectral information. Chemical characterization of membrane receptors in intact cellular membranes is important for understanding membrane signaling and drug targeting. These results provide a new method to investigate the chemical interactions associated with ligand binding to membrane receptors in cells.     

The Mechanism of Ligand‐Induced Activation or Inhibition of μ‐ and κ‐Opioid Receptors

G‐protein‐coupled receptors (GPCRs) are important targets for treating severe diseases. However why certain molecules act as activators whereas others, with similar structures, block GPCR activation, is poorly understood since the same molecule can activate one receptor subtype while blocking another closely related receptor. To shed light on these central questions, we used all‐atom, long‐time‐scale molecular dynamics simulations on the κ‐opioid and μ‐opioid receptors (κOR and μOR). We found that water molecules penetrating into the receptor interior mediate the activating versus blocking effects of a particular ligand–receptor interaction. Both the size and the flexibility of the bound ligand regulated water influx into the receptor. The solvent‐accessible inner surface area was found to be a parameter that can help predict the function of the bound ligand.     

The Effect of Ligand Mobility on the Cellular Interaction of Multivalent Nanoparticles

Multivalent nanoparticle binding to cells can be of picomolar avidity making such interactions almost as intense as those seen with antibodies. However, reducing nanoparticle design exclusively to avidity optimization by the choice of ligand and its surface density does not sufficiently account for controlling and understanding cell–particle interactions. Cell uptake, for example, is of paramount significance for a plethora of biomedical applications and does not exclusively depend on the intensity of multivalency. In this study, it is shown that the mobility of ligands tethered to particle surfaces has a substantial impact on particle fate upon binding. Nanoparticles carrying angiotensin‐II tethered to highly mobile 5 kDa long poly(ethylene glycol) (PEG) chains separated by ligand‐free 2 kDa short PEG chains show a superior accumulation in angiotensin‐II receptor type 1 positive cells. In contrast, when ligand mobility is constrained by densely packing the nanoparticle surface with 5 kDa PEG chains only, cell uptake decreases by 50%. Remarkably, irrespective of ligand mobility and density both particle types have similar EC50 values in the 1–3 × 10^?9 m range. These findings demonstrate that ligand mobility on the nanoparticle corona is an indispensable attribute to be considered in particle design to achieve optimal cell uptake via multivalent interactions.     

AquaBridge: A novel method for systematic search of structural water molecules within the protein active sites

We have developed a novel method for calculation of the water bridges that can be formed in the active sites of proteins in the absence or in the presence of small‐molecule ligands. We tested its efficiency on a representative set of human ATP‐binding proteins, and show that the docking accuracy of ligands can be substantially improved when water bridges are included in the modeling of protein–ligand interactions. Our analysis of binding pocket hydration can be a useful addition to the in silico approaches of Drug Design. © 2015 Wiley Periodicals, Inc.     

Cell‐Derived Vesicles for Single‐Molecule Imaging of Membrane Proteins

A new approach is presented for the application of single‐molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single‐molecule measurements. Cell‐derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution‐based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single‐molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single‐molecule studies to previously inaccessible classes of receptors.     

Kinetics of ligand binding to membrane receptors from equilibrium fluctuation analysis of single binding events

Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.     

Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling

Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell surface. In this study, transferrin receptor 1 (TfR1) was fused to peptide carrier protein (PCP), and the TfR1-PCP fusion protein was specifically labeled with fluorophore Alexa 488 by Sfp. The trafficking of transferrin-TfR1-PCP complex during the process of transferrin-mediated iron uptake was imaged by fluorescence resonance energy transfer between the fluorescently labeled transferrin ligand and TfR1 receptor. We thus demonstrated that Sfp-catalyzed small molecule labeling of the PCP tag represents a practical and efficient tool for molecular imaging studies in living cells.     

Reversibly Triggered Protein–Ligand Assemblies in Giant Vesicles

External small‐molecule triggers were used to reversibly control dynamic protein–ligand interactions in giant vesicles. An alcohol dehydrogenase was employed to increase or decrease the interior pH upon conversion of two different small‐molecule substrates, thereby modulating the pH‐sensitive interaction between a Ni‐NTA ligand on the vesicle membrane and an oligohistidine‐tagged protein in the lumen. By alternating the small‐molecule substrates the interaction could be reversed.     

Selective and Wash‐Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single‐Molecule Imaging of μ‐Opioid Receptor Dimerization

μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level.     

Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single‐Molecule Tracking Study

We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper‐free click chemistry. Finally, these biotin‐tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin‐induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA‐protein networks employing single‐molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface.     

Rotational and Translational Dynamics of Ras Proteins upon Binding to Model Membrane Systems

Plasma-membrane-associated Ras proteins typically control signal transduction processes. As nanoclustering and membrane viscosity sensing provide plausible signaling mechanisms, determination of the rotational and translational dynamics of membrane-bound Ras isoforms can help to link their dynamic mobility to their function. Herein, by using time-resolved fluorescence anisotropy and correlation spectroscopic measurements, we obtain the rotational-correlation time and the translational diffusion coefficient of lipidated boron-dipyrromethene-labeled Ras, both in bulk Ras and upon membrane binding. The results show that the second lipidation motif of N-Ras triggers dimer formation in bulk solution, whereas K-Ras4B is monomeric. Upon membrane binding, an essentially free rotation of the G-domain is observed, along with a high lateral mobility; the latter is essentially limited by the viscosity of the membrane and by lipid-mediated electrostatic interactions. This high diffusional mobility warrants rapid recognition–binding sequences in the membrane-bound state, thereby facilitating efficient interactions between the Ras proteins and scaffolding or effector proteins. The lipid-like rapid lateral diffusion observed here complies with in vivo data.     

PI by NMR: Probing CH–π Interactions in Protein–Ligand Complexes by NMR Spectroscopy

While CH–π interactions with target proteins are crucial determinants for the affinity of arguably every drug molecule, no method exists to directly measure the strength of individual CH–π interactions in drug–protein complexes. Herein, we present a fast and reliable methodology called PI (π interactions) by NMR, which can differentiate the strength of protein–ligand CH–π interactions in solution. By combining selective amino‐acid side‐chain labeling with ¹H‐¹³C NMR, we are able to identify specific protein protons of side‐chains engaged in CH–π interactions with aromatic ring systems of a ligand, based solely on ¹H chemical‐shift values of the interacting protein aromatic ring protons. The information encoded in the chemical shifts induced by such interactions serves as a proxy for the strength of each individual CH–π interaction. PI by NMR changes the paradigm by which chemists can optimize the potency of drug candidates: direct determination of individual π interactions rather than averaged measures of all interactions.     

Fine Tuning of Chlorophyll Spectra by Protein‐Induced Ring Deformation

The ability to tune the light‐absorption properties of chlorophylls by their protein environment is the key to the robustness and high efficiency of photosynthetic light‐harvesting proteins. Unfortunately, the intricacy of the natural complexes makes it very difficult to identify and isolate specific protein–pigment interactions that underlie the spectral‐tuning mechanisms. Herein we identify and demonstrate the tuning mechanism of chlorophyll spectra in type II water‐soluble chlorophyll binding proteins from Brassicaceae (WSCPs). By comparing the molecular structures of two natural WSCPs we correlate a shift in the chlorophyll red absorption band with deformation of its tetrapyrrole macrocycle that is induced by changing the position of a nearby tryptophan residue. We show by a set of reciprocal point mutations that this change accounts for up to 2/3 of the observed spectral shift between the two natural variants.     

An NMR Method To Pinpoint Supramolecular Ligand Binding to Basic Residues on Proteins

Targeting protein surfaces involved in protein–protein interactions by using supramolecular chemistry is a rapidly growing field. NMR spectroscopy is the method of choice to map ligand‐binding sites with single‐residue resolution by amide chemical shift perturbation and line broadening. However, large aromatic ligands affect NMR signals over a greater distance, and the binding site cannot be determined unambiguously by relying on backbone signals only. We herein employed Lys‐ and Arg‐specific H2(C)N NMR experiments to directly observe the side‐chain atoms in close contact with the ligand, for which the largest changes in the NMR signals are expected. The binding of Lys‐ and Arg‐specific supramolecular tweezers and a calixarene to two model proteins was studied. The H2(C)N spectra track the terminal CH₂ groups of all Lys and Arg residues, revealing significant differences in their binding kinetics and chemical shift perturbation, and can be used to clearly pinpoint the order of ligand binding.