Temperature Dependency of CLC-ec1 Dimerization in Lipid Bilayers

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

BackgroundFörster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (kD→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.Methodology/Principal FindingsThe generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.Conclusions/SignificanceWe conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.

The catalytic activity of mitogen-activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using invitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live-cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss-of-function double-mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl-induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22-induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.

G-protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase with an important function in the desensitization of G-protein-coupled receptors. Based on its ability to bind G-protein βγ subunits as well as activated Gαq subunits, it can be considered as an effector for G-proteins. The recruitment of GRK2 to activated receptors is well known to be mediated by Gβγ together with negatively charged membrane phospholipids. In the current study, we address the role of Gαq on the interaction of GRK2 with activated Gq-protein-coupled receptors. Therefore, we established new Förster resonance energy transfer (FRET)-based assays to study the interaction of GRK2 with the M3-acetylcholine (M3-ACh) receptor as well as Gq-protein subunits with high spatiotemporal resolution in single living human embryonic kidney 293T cells. M3-ACh receptor stimulation with 10 µM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding affinity toward Gαq [GRK2(D110A)] and Gβγ [GRK2(R587Q)] were used to determine the specific role of Gq-protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Gαq enhances the extent and stability of the GRK2-M3-ACh receptor interaction, and that not only Gβγ but also Gαq can target GRK2 to the membrane. This reveals an important role of Gαq in efficient recruitment of GRK2 to M3-ACh receptors. Furthermore, interactions between Gαq and GRK2 were associated with a prolongation of the interaction between GRK2 and the M3-ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Gαq influences signaling and desensitization.

Polyethylene glycol-phospholipid micelles form a major class of nanocarriers in pharmacy and medicine due to proven capability in drug solubilization, sustained drug release, and evidence for targeted drug delivery in vivo. In this report, we have prepared micelles composed of PEG-block-poly(N-hexyl stearate l-aspartamide) (PEG-b-PHSA), having nine stearic acid side chains, and have studied their stability in the presence of serum proteins by Forster resonance energy transfer (FRET) experiments. In the presence of serum albumin, alpha and beta globulins, or gamma globulins, there are minimal changes in FRET over two hours in vitro, indicating integrity of PEG-b-PHSA micelles. In contrast, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (PEG-DSPE) micelles lose FRET over two hours in vitro, especially in the presence of alpha and beta globulins, indicating the disruption of PEG-DSPE micelles and leakage of fluorescent probes. Owing to the aliphatic nature of DSPE and PHSA, both PEG-b-PHSA and PEG-DSPE micelles efficiently solubilize amphotericin B (AmB), a poorly water-soluble antifungal agent used to combat systemic mycoses. However, only PEG-b-PHSA micelles gradually liberate AmB in the presence of alpha and beta globulins, based on time-dependent changes in the self-aggregation state of AmB, monitored by UV/vis spectroscopy. PEG-b-PHSA micelles are remarkably stable in the presence of serum proteins and a more stable alternative for poorly water-soluble drugs, which have been solubilized by PEG-DSPE micelles.

Capillary electrophoretic studies on displacement and proteolytic cleavage of surface bound oligohistidine peptide on quantum dots

Amphiphilic block copolymers with weak polyelectrolyte blocks can assemble stimulus-responsive nanostructures and interfaces. Applications of these materials in drug delivery, biomimetics, and sensing largely rely on the well-understood swelling of polyelectrolyte chains upon deprotonation, often induced by changes in pH or ionic strength. This deprotonation can also tune interfacial interactions between the polyelectrolyte blocks and surrounding solution, an effect which is less studied than morphological swelling of polyelectrolytes but can be just as critical for intended function. Here, we investigate whether the pH-driven morphological response of polyelectrolyte-bearing nanostructures also affects the interactions of these nanostructures with molecules in solution, using micelles of a short-chain polybutadiene-block-poly(acrylic acid) (pBd-pAA) as a model system. We introduce a Förster resonance energy transfer (FRET) approach to probe interactions between micelles and fluorescent molecular solutes as a function of solution pH. As expected, the pAA corona of these pBd-pAA micelles increases in thickness monotonically as a function of pH. However, FRET efficiency, which provides a metric of the spatial proximity of fluorescently labeled micelles and freely diffusing fluorophores, exhibits complex nonmonotonic behavior as a function of pH, indicating that the average separation of micelles and acceptor fluorophores is not strictly correlated with micelle swelling. Dialysis experiments quantify the affinity of fluorophores for micelles as a function of pH, confirming that changes in FRET are driven almost entirely by the pH-dependent affinity of the pAA block for the investigated molecular fluorophores, not simply by a shape change of the pAA corona. This study provides key insights into the interfacial interactions between weak-polyelectrolyte-bearing nanostructures and molecular solutes, of importance for the development of their stimulus-responsive applications.

Interaction of a two-transmembrane-helix peptide with lipid bilayers and dodecyl sulfate micelles

Membrane Protein Dimerization in Cell-Derived Lipid Membranes Measured by FRET with MC Simulations

Herein, an ionic material (IM) with Förster Resonance Energy Transfer (FRET) characteristics is reported for the first time. The IM is designed by pairing a Nile Blue A cation (NBA+) with an anionic near-infrared (NIR) dye, IR820−, using a facile ion exchange reaction. These two dyes absorb at different wavelength regions. In addition, NBA+ fluorescence emission spectrum overlaps with IR820− absorption spectrum, which is one requirement for the occurrence of the FRET phenomenon. Therefore, the photophysical properties of the IM were studied in detail to investigate the FRET mechanism in IM for potential dye sensitized solar cell (DSSCs) application. Detailed examination of photophysical properties of parent compounds, a mixture of the parent compounds, and the IM revealed that the IM exhibits FRET characteristics, but not the mixture of two dyes. The presence of spectator counterion in the mixture hindered the FRET mechanism while in the IM, both dyes are in close proximity as an ion pair, thus exhibiting FRET. All FRET parameters such as spectral overlap integral, Förster distance, and FRET energy confirm the FRET characteristics of the IM. This article presents a simple synthesis of a compound with FRET properties which can be further used for a variety of applications.

The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and Experiments

Live-cell FLIM-FRET using a commercially available system.

Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane

Fӧrster resonance energy transfer (FRET) based cAMP sensors have become a standard approach to visualize cAMP signals in cells. However, FRET sensors inherently offer low signal-to-noise ratios, making cAMP measurements challenging, especially in 3 spatial dimensions. In previous studies, we used 4-dimensional (x, y, z, and λ) hyperspectral imaging and analysis approaches to measure agonist – induced distribution of cAMP signals in cultured pulmonary microvascular endothelial cells. In many cell types including PMVECs, transient transfection of FRET sensors results in a low transfection efficiency (1-5%), which limits the possibilities of studying spatial distribution of FRET signals in many cells simultaneously. We developed a transgenic rat model that expresses the H187 FRET sensor. In current studies, we utilize hyperspectral imaging and analysis approaches to characterize the distribution of FRET signals in different tissues and vessels harvested from transgenic rats. Hyperspectral z-stacks of tissues and vessels were acquired using a Nikon A1R confocal microscope equipped with 20X multi-immersion objective and a 32 channel PMT detector. Samples were excited using 405 nm and 561 nm lasers and emission was collected from 424 nm -724 nm at 10 nm intervals. Tissues including lungs, heart, mesentery, kidney, expressed the H187 FRET sensor. In conclusion, transgenic rats provide a platform to visualize cAMP signals in-vitro and in-situ.

We propose a linear measure of the Forster resonant energy transfer (FRET) efficiency derived in joint, donor and acceptor, intensity space and compare it quantitatively with the conventional ratiometric FRET measure normally used for FRET fluorescence microscopy images. Because of the low signal-to-noise characteristic of these images, particularly time-lapse images, we determine a quantitative model of shot noise and combine it with our measure. This provides a single measure of the certainty of FRET events that can be used as a pixel-wise classifier of FRET images. We evaluate our measures quantitatively by simulating photon emission under realistic conditions and demonstrate: linearity with FRET efficiency; less bias to shot noise than the ratio; and high correlation (r = 0.96) between our FRET certainty measure and SNR.