Acceptor Fluorophores Research Articles

Abstract 12676: C-Terminal Residues of Phospholamban are Critical Determinants of the Quaternary Structure and Function of the Calcium ATPase Regulatory Complex

Rationale: A naturally occurring missense Leu-39stop (L39X) mutation in phospholamban (PLB) results in truncation of the C-terminal transmembrane domain, leading to cardiomyopathy and premature death in humans. Objective: The goal of this study was to determine the structural and regulatory role of the C-terminal residues of PLB in the membranes of living cells. Methods and Results: We fused fluorescent protein tags to PLB and cardiac Ca 2+ ATPase (SERCA) to investigate the role of PLB C-terminal residues for membrane localization, PLB oligomerization and SERCA regulation. Alanine substitution of C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB. Notably, substitution mutation V49A had profound effects on pentamer structure and regulatory complex conformation, increasing and decreasing probe separation distance, respectively. Progressive deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. Selective permeabilization of the plasma membrane by saponin resulted in diffusion of fluorescently labeled PLB out of the cells, consistent with solubilization of truncated proteins. Molecular dynamics simulations recapitulated decreased bilayer anchoring for truncated PLB. C-terminal truncations resulted in progressive loss of PLB-PLB FRET, due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the SERCA-PLB binding affinity, and loss of inhibitory potency as quantified by Ca 2+ -dependent ATPase activity. However, despite decreased SERCA-PLB binding, intermolecular FRET was paradoxically increased as a result of a 14.5 Å decrease in the distance between donor and acceptor fluorophores. Conclusions: We conclude that PLB C-terminal residues are critical for membrane anchoring and quaternary structure determination of PLB pentamer and PLB-SERCA regulatory complex. The loss of membrane registration restraint by C-terminal residues (especially V49) causes displacement of PLB to an alternative position on SERCA. The data are compatible with a model in which PLB binds to the canonical inhibitory binding site and an additional novel site.

One‐pot preparation of multicolor polymeric nanoparticles with high brightness by single wavelength excitation

ABSTRACTFluorescent nanoparticles with multiplex distinct emission signatures and high brightness by a single wavelength excitation are substantially needed in multiplex bioassays and imaging. In this study, we synthesized fluorescent polymeric nanoparticles incorporated with three polymerizable organic dyes via a one‐pot miniemulsion. By altering the doping ratio of three tandem dyes, the nanoparticles display abundant multiple fluorescence such as blue, cyan, green, orange, pink, red etc., together with distinguishable emission signatures under a single wavelength excitation, which were arising from the effective fluorescence resonance energy transfer (FRET) between the three energy‐matched dyes. Meanwhile, a large Stokes shift (up to 250 nm) can be generated by taking place multiple FRET cascade mechanism between donor and acceptor fluorophores in nanoparticles, which also suggests broad applications in biological labeling and imaging. Moreover, these nanoparticles are uniform in size, highly bright, excellently photostable, and shown prominent longterm stability. Overall, the novel multicolor fluorescent polymeric nanoparticles augur well for their potential applications in multiplexed bioanalysis and emitting displays. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41492.

Modest Influence of FRET Chromophores on the Properties of Unfolded Proteins

Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ2, has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances.

FRET: signals hidden within the noise.

F€ orster resonance energy transfer (FRET) is a physical phenomenon that has been of incredible utility to the scientific community, enabling the study of a wide range of nanoscale, distance-dependent measurements. FRET occurs when energy is nonradiatively transferred from an excited fluorophore (the donor fluorophore) to a second fluorophore in near proximity (the acceptor fluorophore) (1,2). Because the rate at which energy is transferred from the donor to the acceptor fluorophore depends on the intermolecular distance raised to the sixth power, FRET provides a highly sensitive measure of intermolecular spacing (2,3). The key parameter in FRET measurements is the FRET efficiency. FRET efficiency is most often described as the ratio of the number of quantized energy events (sometimes referred to as virtual photons (4)) transferred from the donor fluorophore to the acceptor fluorophore, divided by the total number of quantized energy events (photons) absorbed by the donor fluorophore (5). Using appropriate measurement and computational techniques, FRET efficiency represents a quantifiable value describing the intermolecular spacing and orientation within a sample. For fluorophores with fixed intermolecular spacing, the value of the measured FRET efficiency has been shown to be relatively constant, regardless of the measurement technique used (6). Hence, FRET reporters can be used to provide absolute quantitative data describing interand intramolecular interactions. Computing the FRET efficiency, whether in fluorescence microscopy or flow cytometry measurements, typically requires a ratiometric calculation (7). Because FRET efficiencies often range from 20 to 60% for biological reporters, the fluorescence emission from the acceptor fluorophore, when excited via energy transfer, usually provides lower signal strength than measurements of acceptor fluorophore emission resulting from direct excitation. Performing ratiometric calculations using relatively weak signals as the inputs can result in compounding error propagation (8). Thus, while FRET, as a tool, offers the ability to probe molecular distances within images or flow cytometry data, it also introduces substantial limitations in sensitivity and variance, due to low signal strength. Low signal strength may also limit spatial and temporal resolution. When performing FRET experiments, trade-offs must usually be made between signal strength (accuracy of the FRET efficiency calculation), spatial resolution, and temporal

Luminescence resonance energy transfer to study conformational changes in membrane proteins expressed in mammalian cells.

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 Å. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

Magnetic Field Effects in Fluorescence of Exciplex and Fluorophore for the Weller Schemes I and II: Similarities and Differences

In exciplex-forming donor–acceptor systems the photoexcited acceptor state can be quenched either by exciplex formation at the contact of the acceptor and the donor, followed by reversible dissociation to the radical-ion pair (Scheme I), or by distant electron transfer yielding a radical-ion pair that can reversibly recombine to produce an exciplex (Scheme II). The fluorescence of the photoexcited electron acceptor (fluorophore) and the exciplex is known to be sensitive to the magnetic field, assisting spin conversion in the resulting geminate radical-ion pair (RIP). An increase of the fluorescence yield in high magnetic fields compared to low fields is known as the magnetic field effect (MFE). In this paper theories of the MFE on both the fluorophore and the exciplex within the Schemes I and II are developed. Kinetics of the reversible reactions are described in terms of the integral encounter theory that unlike the differential encounter theory is capable of dealing with such reactions. Regions of the parameters where MFE is observable are discussed. Comparison of functional dependencies of MFE on the model parameters for the Schemes I and II is carried out. Magnitudes and dependencies of MFE on the model parameters are found to be very similar for the Schemes I and II, except the dependencies of the fluorophore and exciplex MFEs on the rate constant of exciplex production from the radical-ion pair, which appear to be opposite for the Schemes I and II.

A protein L-based immunodiagnostic approach utilizing time-resolved Förster resonance energy transfer.

Chelated lanthanides such as europium (Eu) have uniquely long fluorescence emission half-lives permitting their use in time-resolved fluorescence (TRF) assays. In Förster resonance energy transfer (FRET) a donor fluorophore transfers its emission energy to an acceptor fluorophore if in sufficiently close proximity. The use of time-resolved (TR) FRET minimizes the autofluorescence of molecules present in biological samples. In this report, we describe a homogenous immunoassay prototype utilizing TR-FRET for detection of antibodies in solution. The assay is based on labeled protein L, a bacterial protein that binds to immunoglobulin (Ig) light chain, and labeled antigen, which upon association with the same Ig molecule produce a TR-FRET active complex. We show that the approach is functional and can be utilized for both mono- and polyvalent antigens. We also compare the assay performance to that of another homogenous TR-FRET immunoassay reported earlier. This novel assay may have wide utility in infectious disease point-of-care diagnostics.

Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val(49)-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.

Contribution of Fluorophore Dynamics and Solvation to Resonant Energy Transfer in Protein-DNA Complexes: A Molecular-Dynamics Study

In Förster resonance energy transfer (FRET) experiments, extracting accurate structural information about macromolecules depends on knowing the positions and orientations of donor and acceptor fluorophores. Several approaches have been employed to reduce uncertainties in quantitative FRET distance measurements. Fluorophore-position distributions can be estimated by surface accessibility (SA) calculations, which compute the region of space explored by the fluorophore within a static macromolecular structure. However, SA models generally do not take fluorophore shape, dye transition-moment orientation, or dye-specific chemical interactions into account. We present a detailed molecular-dynamics (MD) treatment of fluorophore dynamics for an ATTO donor/acceptor dye pair and specifically consider as case studies dye-labeled protein-DNA intermediates in Cre site-specific recombination. We carried out MD simulations in both an aqueous solution and glycerol/water mixtures to assess the effects of experimental solvent systems on dye dynamics. Our results unequivocally show that MD simulations capture solvent effects and dye-dye interactions that can dramatically affect energy transfer efficiency. We also show that results from SA models and MD simulations strongly diverge in cases where donor and acceptor fluorophores are in close proximity. Although atomistic simulations are computationally more expensive than SA models, explicit MD studies are likely to give more realistic results in both homogeneous and mixed solvents. Our study underscores the model-dependent nature of FRET analyses, but also provides a starting point to develop more realistic in silico approaches for obtaining experimental ensemble and single-molecule FRET data.

Issue Cover (August 2014)

Cover legend: Dynamin oligomerization in cells detected by fluorescence lifetime imaging microscopy. Cos-7 cells expressing dynamin were treated with 30 μM Bis-T-23 (small molecule) which induces dynamin oligomerization into rings. After 20 min, cells were fixed and stained using monoclonal anti-GTPase antibody conjugated with Alexa Fluor 488 (donor fluorophore) and monoclonal anti-PRD antibody conjugated with Alexa Fluor 594 (acceptor fluorophore). Lifetimes are shown as discrete colors: fast lifetimes between 0–2200 ps represent ≥20% fluorescence resonance energy transfer (FRET) efficiency and are shown in red, whereas slow lifetimes above 2201 ps are shown in blue (no FRET). Positive FRET signal was only detected when dynamin oligomerized. In COS-7 cells, dynamin oligomers (positive signal) were detected throughout the cytoplasm, at the transition zone in which cortical actin transitions to stress fibers, and often at the cytoplasmic face of the nuclei. See Gu et al. Traffic 15(8): 819–838. Read the full article on doi:10.1111/tra.12178

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

The hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA-dependent RNA polymerase that is essentially required for viral replication. Although previous studies revealed important properties of static NS5B-RNA complexes, the nature and relevance of dynamic interactions have yet to be elucidated. Here, we devised a single molecule Förster Resonance Energy Transfer (SM-FRET) assay to monitor temporal changes upon binding of NS5B to surface immobilized RNA templates. The data show enzyme association-dissociation events that occur within the time resolution of our setup as well as FRET-fluctuations in association with stable binary complexes that extend over prolonged periods of time. Fluctuations are shown to be dependent on the length of the RNA substrate, and enzyme concentration. Mutations in close proximity to the template entrance (K98E, K100E), and in the center of the RNA binding channel (R394E), reduce both the population of RNA-bound enzyme and the fluctuations associated to the binary complex. Similar observations are reported with an allosteric nonnucleoside NS5B inhibitor. Our assay enables for the first time the visualization of association-dissociation events of HCV-NS5B with RNA, and also the direct monitoring of the interaction between HCV NS5B, its RNA template, and finger loop inhibitors. We observe both a remarkably low dissociation rate for wild type HCV NS5B, and a highly dynamic enzyme-RNA binary complex. These results provide a plausible mechanism for formation of a productive binary NS5B-RNA complex, here NS5B slides along the RNA template facilitating positioning of its 3' terminus at the enzyme active site.

Collapse of a long axis: single-molecule Förster resonance energy transfer and serpin equilibrium unfolding.

The energy required for mechanical inhibition of target proteases is stored in the native structure of inhibitory serpins and accessed by serpin structural remodeling. The overall serpin fold is ellipsoidal with one long and two short axes. Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms. This suggests that ellipticity, as typified by the long axis, may be important for both function and folding. Placement of donor and acceptor fluorophores approximately along the long axis or one of the short axes allows single-pair Förster resonance energy transfer (spFRET) to report on both unfolding transitions and the time-averaged shape of different conformations. Equilibrium unfolding and refolding studies of the well-characterized inhibitory serpin α1-antitrypsin reveal that the long axis collapses in the folding intermediates while the monitored short axis expands. These energetically distinct intermediates are thus more spherical than the native state. Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations. This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins.

Development of an AP-FRET based analysis for characterizing RNA-protein interactions in myotonic dystrophy (DM1).

Förster Resonance Energy Transfer (FRET) microscopy is a powerful tool used to identify molecular interactions in live or fixed cells using a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity. FRET can be a very sensitive tool to study protein-protein and/or protein-nucleic acids interactions. RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy. Myotonic dystrophy (DM1) is caused by a (CTG)n repeat expansion in the 3′ UTR of the DMPK gene which results in nuclear retention of mutant DMPK transcripts in RNA foci. This results in toxic gain-of-function effects mediated through altered functions of RNA-binding proteins (e.g. MBNL1, hnRNPH, CUGBP1). In this study we demonstrate the potential of a new acceptor photobleaching assay to measure FRET (AP-FRET) between RNA and protein. We chose to focus on the interaction between MBNL1 and mutant DMPK mRNA in cells from DM1 patients due to the strong microscopic evidence of their co-localization. Using this technique we have direct evidence of intracellular interaction between MBNL1 and the DMPK RNA. Furthermore using the AP-FRET assay and MBNL1 mutants, we show that all four zinc-finger motifs in MBNL1 are crucial for MBNL1-RNA foci interactions. The data derived using this new assay provides compelling evidence for the interaction between RNA binding proteins and RNA foci, and mechanistic insights into MBNL1-RNA foci interaction demonstrating the power of AP-FRET in examining RNA-Protein interactions in DM1.

Homogeneous FRET immunoassays for use in personalized medicine

IntroductionDrug replacement and maintenance therapy of methadone has been used all over the world in maintenance treatment programmes of opioid dependence. The effectiveness of drug maintenance therapy in a patient is highly depended on the drug levels in the patient's blood. A popular technique in drug monitoring is competitive immunoassay for example ELISA which has proven to be simpler and less expensive. However, multiple processing steps are still required to achieve the required sensitivity. In order to increase the efficiency for detection of drugs, homogeneous immunoassays that do not require any time-consuming washing and separation steps have an advantage. ObjectiveTo develop a one-step homogeneous FRET immunoassay against methadone for use in personalized medicine. MethodsFRET based homogeneous immunoassay against methadone was designed by using fluorescein isothiocyanide – Lissamine™ Rhodamine B Ethylenediamine as donor fluorophore and acceptor fluorophore. These organic dye molecules were then conjugated with the monoclonal antibodies which against methadone and the antigen (methadone). The measurement of the signal of this FRET assay was performed after the mixing of this donor-acceptor pair. Then, the percentage of energy transfer was calculated and compared. The optimum conditions and reproducibility for this assay was determined in this study. Results & DiscussionFRET based immunoassay that was developed from this study is highly reproducible. This method was proven to be a very rapid method for quantitative detection of methadone. ConclusionFRET based immunoassay can be used as suitable alternative for the monitoring of MMT serum levels.

Determining Acceptor Dye Quantum Yield from Pulsed Interleaved Single-Pair FRET Measurements

Single-pair fluorescence resonant energy transfer (spFRET) is a widely accepted technique for determining intra/inter molecular distances on nanometer scale. We present spFRET measurements of freely diffusing DNA constructs using pulsed interleaved excitation (PIE). We engineered a FRET system consisting of two complementary, single stranded DNA oligonucleotides containing either a donor or acceptor fluorophore. The FRET donor was attached to the antisense strand at 12 different positions, each on separate strands. The oligonucleotide containing the FRET acceptor was the sense strand. The two strands were annealed together providing incremental distance changes from 5 to 27 base pairs, by moving the donor fluorophore along the antisense strand every other base pair. We used PIE to sort the species into donor-only, donor-acceptor and acceptor-only species. Average burst intensity from the donor-acceptor population that has been directly excited by the red laser provides a relative measure of the quantum yield of the acceptor dye. Recovery of the quantum yield of the acceptor affords reliable estimates of transfer efficiency, hence inter-dye distances. We find that PIE-mediated sorting produces reliable transfer efficiencies from the donor-acceptor population in the presence of a 20-fold excess of donor-only species.

Tmfret to Study Short-Range Interactions at the Membrane

Fluorescence resonance energy transfer (FRET) is a technique used to determine the distance between two fluorophores. However, the precision of the distance measurement is limited by the size of the fluorophores and their linkers. In addition, distances over which traditional FRET pairs are sensitive is on the order of the size of many proteins. Thus, short-range interactions have been difficult to study with FRET.Transition Metal Ion FRET (tmFRET) is a novel technique in which the acceptor fluorophore is replaced with a transition metal divalent cation which acts as a non-fluorescent acceptor. The proximity of a fluorescently labeled protein to the metal ions can be determined by the quenching of the donor fluorophore. The small size of the metal ions coupled with the use of small donor fluorphores, such as bimane, allows precise measurement of short-range interactions on the order of 8-20 A.Here we studied the use of tmFRET for measuring interactions at a cell membrane. As proof-of-principle, we used the membrane-resident dye Octadecyl Rhodamine B Chloride (R18) as a FRET donor and metal-bound stearoyl-NTA as a non-fluorescent quencher. Because stearoyl-NTA and R18 both reside in the membrane and have the same acyl chains, we expect the metal-bound NTA group to be well within the distance range of R18 required for tmFRET. Cells were labeled with R18 via transient introduction into the bath. Perfusion of metal-bound stearoyl-NTA into the bath produced quenching of the R18 fluorescence. We studied the reversibility of this effect by subsequently perfusing the cells with EDTA, which chelates the metal ions. Finally, we determined the concentration dependence of R18 quenching by metal-bound stearoyl-NTA. These experiments lay the groundwork for future studies in which tmFRET will be used to measure the distance of fluorophore-tagged proteins from the membrane.

Phospholamban C-Terminal Truncations Including Heart Failure Mutation L39Stop Decrease Membrane Localization and Oligomerization and Alter the Structure of the PLB-Serca Complex

A naturally occurring missense Leu-39stop (L39X) mutation in phospholamban (PLB) results in truncation of the C-terminal transmembrane domain, leading to cardiomyopathy and premature death. In this study, we fused PLB and SERCA to fluorescent protein tags to determine the structural and thermodynamic consequences of progressive truncations of the C-terminal residues of PLB in the membranes of living cells. We found that deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. Selective permeabilization of the plasma membrane by saponin resulted in diffusion of fluorescently labeled PLB out of the cells, consistent with solubilization of truncated proteins. Western blot analysis showed the expected mobilities for truncation mutants relative to full length PLB-WT, indicating that the observed solubilization of PLB truncation mutants is not due to proteolysis. Moreover, molecular dynamics simulations recapitulated the observed loss of stable bilayer anchoring for truncated PLB. Fluorescence resonance energy transfer (FRET) analysis revealed that C-terminal truncations resulted in progressive loss of PLB-PLB FRET, indicating a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the SERCA-PLB binding affinity. Despite this decrease in affinity, SERCA-PLB FRET was paradoxically increased by deletion of up to 4 C-terminal residues as a result of a 14 angstrom decrease in the distance between donor and acceptor fluorophores. Truncation of PLB also decreased its inhibitory potency as quantified by calcium-dependent ATPase activity. We conclude that the C-terminal residues are critical for PLB localization, SERCA-PLB regulatory complex conformation, and PLB regulatory function.

Structural Dynamics Underlying Pip2 Modulation of Kir Channels Revealed by Single Molecule FRET

Ion channels are gated by a multitude of electrical, chemical and mechanical stimuli, and thereby play key roles in many physiological processes. Static crystal structures and dynamic electrophysiological studies have generated invaluable molecular insights into channel gating, but the time trajectory of conformational changes has been unattainable. KirBac1.1 is a model for one of the three main K channel families. In the present study, we have successfully (1) labeled purified tetrameric protein molecules with single FRET donor and acceptor fluorophore pairs at specific locations; (2) functionally reconstituted the proteins into liposomes; (3) examined the dynamics of channel structure in liposomes with single molecule FRET techniques. Our results indicate that the amphipathic 'slide helices' of KirBac1.1 that surround the channel gate immediately below the membrane, move toward each other to narrow the pore in the presence of PIP2, which closes the channel; the cytoplasmic domain resides at two major structural states, but demonstrates an overall shift away from the pore axis upon PIP2 inhibition; the extracellular loop of KirBac1.1, adjacent to the selectivity filter, is structurally rigid and does not move during PIP2 gating. Both slide helix and cytoplasmic domain structures become less dynamic when the channel is inhibited by PIP2, which implies that PIP2 acts as a 'lock' to confine the structural fluctuations of the channel protein. We further probed the relative motions of the KirBac1.1 transmembrane domain versus the cytoplasmic domain. The results indicate that the entire cytoplasmic domain moves away from the transmembrane domain during PIP2 inhibition, which may result from a rigid body motion coupled to TM2 bending. In summary, we provide direct observations of dynamic structures of an ion channel within a lipid membrane environment, revealing dynamic structural rearrangements KirBac1.1 upon PIP2 inhibition.

Intra- and Interlayer Energy Transfer in Planar Systems Based on Amphiphilic Naphthalimide Derivatives

The present work considers the possibility of increasing the information signal of thin-film sensitive element of the sensor based on amphiphilic crown substituted derivative of 4-(acylamino)-1,8-naphthalimide via employment of resonance photoexcitation energy transfer process. In such system, information signal is the fluorescence of acceptor fluorophore (4-alkylamino-1,8-naphthalimide), which is excited upon non-radiative transfer of the energy from excited donor fluorophore (crown containing derivative of 4-(acylamino)-1,8-naphthalimide), crown ether fragment of which is “occupied” by coordination interaction with specific cation and thus does not participate in rapid relaxation of the excitation by the photoinduced electron transfer mechanism. For realization of this goal, the synthesis of novel amphiphilic naphthalimide derivatives is carried out; conditions for obtaining of stable monolayers and Langmuir-Blodgett films based on them are studied. It is established that azadithiacrown substituted amphiphilic fluoroionophore in monolayers at air/mercury and silver perchlorate aqueous solutions interface retains the ability for efficient binding of Ag + and Hg 2+ known for its solutions. Described principle of generation of fluorescent signal using mixed monolayer containing the donor-acceptor couple allows one to increase the value of optical response of the monolayer to the presence of assessed cations by 2.5 times. However, the monolayer fluorescence intensity remains too low for practical application. In order to optimize the conditions of the energy transfer process in more practically important solid-state sensory elements we studied the mechanism of non-radiative photoexcitation energy transfer in the multilayer thin-film system based on amphiphilic naphthalimide derivatives obtained by Langmuir-Blodgett (LB) technique. It is shown that this problem can be solved by regulation of the distance between donor and acceptor layers of LBF. Optimal thickness of the separating layer of stearic acid, which for this system amounted to 7 nm, was determined experimentally. Obtained results open up new perspectives for increase of the information signal reading efficiency, which should find application in development of sensitive elements of sensor devices.

Lanthanides and Quantum Dots as Förster Resonance Energy Transfer Agents for Diagnostics and Cellular Imaging

Luminescent lanthanide labels (LLLs) and semiconductor quantum dots (QDs) are two very special classes of (at least partially) inorganic fluorophores, which provide unique properties for Förster resonance energy transfer (FRET). FRET is an energy-transfer process between an excited donor fluorophore and a ground-state acceptor fluorophore in close proximity (approximately 1-20 nm), and therefore it is extremely well suited for biosensing applications in optical spectroscopy and microscopy. Within this cogent review, we will outline the main photophysical advantages of LLLs and QDs and their special properties for FRET. We will then focus on some recent applications from the FRET biosensing literature using LLLs as donors and QDs as donors and acceptors in combination with several other fluorophores. Recent examples of combining LLLs and QDs for spectral and temporal multiplexing from single-step to multistep FRET demonstrate the versatile and powerful biosensing capabilities of this unique FRET pair. As this review is published in the Forum on Imaging and Sensing, we will also present some new results of our groups concerning LLL-based time-gated cellular imaging with optically trifunctional antibodies and LLL-to-QD FRET-based homogeneous sandwich immunoassays for the detection of carcinoembryonic antigen.