Intramolecular Fluorescence Resonance Energy Transfer Research Articles

Detection of conformationally changed MBP using intramolecular FRET

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E)=∼0.11, Distance (D)=∼6.93nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.

The Mechanism of CSF-1-induced Wiskott-Aldrich Syndrome Protein Activation in Vivo: A ROLE FOR PHOSPHATIDYLINOSITOL 3-KINASE AND Cdc42

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.

Towards in vivo imaging of intramolecular fluorescence resonance energy transfer parameters

Fluorescence resonance energy transfer (FRET) is a nonradiative energy transfer process based on dipole-dipole interaction between donor and acceptor fluorophores that are spatially separated by a distance of a few nanometers. FRET has proved to be of immense value in the study of cellular function and the underlying cause of disease due to, for example, protein misfolding (of consequence in Alzheimer's disease). The standard parameterization in intramolecular FRET is the lifetime and yield, which can be related to the donor-acceptor (DA) distance. FRET imaging has thus far been limited to in vitro or near-surface microscopy because of the deleterious effects of substantial scatter. We show that it is possible to extract the microscopic FRET parameters in a highly scattering environment by incorporating the FRET kinetics of an ensemble of DA molecules connected by a flexible or rigid linker into an optical diffusion tomography (ODT) framework. We demonstrate the efficacy of our approach for extracting the microscopic DA distance through simulations and an experiment using a phantom with scattering properties similar to tissue. Our method will allow the in vivo imaging of FRET parameters in deep tissue, and hence provide a new vehicle for the fundamental study of disease.

Anomalous Photophysics of H1 Antihistamines in Aqueous Solution

Electronic absorption, emission, and excitation spectra, and fluorescence lifetimes of two H1 antihistamines--tripelennamine and mepyramine--are investigated in detail to ascertain their usefulness as fluorescent probes for ligand binding to G-protein coupled receptors. The photophysical behavior of these compounds in aqueous solution is complex due to the presence of three protonable nitrogens, intramolecular hydrogen bonding, quenching due to the formation of a charge transfer state, and intramolecular fluorescence resonance energy transfer. At physiological pH values, anomalous photophysical behavior is observed: the compounds are found to be in a ground-state equilibrium mixture of two species, one with the alkylamine tail involved in an intramolecular hydrogen bond and a second without such a bond. This internal hydrogen-bonded tail has a profound effect on the ground and excited-state properties of both tripelennamine and mepyramine, which is further elucidated by comparing them to the reference compounds 2-aminopyridine and 2-(N,N-dimethylamino)pyridine.

Mechanical stimulus alters conformation of type 1 parathyroid hormone receptor in bone cells

The molecular mechanisms by which bone cells transduce mechanical stimuli into intracellular biochemical responses have yet to be established. There is evidence that mechanical stimulation acts synergistically with parathyroid hormone PTH(1-34) in mediating bone growth. Using picosecond time-resolved fluorescence microscopy and G protein-coupled receptor conformation-sensitive fluorescence resonance energy transfer (FRET), we investigated conformational transitions in parathyroid hormone type 1 receptor (PTH1R). 1) A genetically engineered PTH1R sensor containing an intramolecular FRET pair was constructed that enabled detection of conformational activity of PTH1R in single cells. 2) The nature of ligand-dependent conformational change of PTH1R depends on the type of ligand: stimulation with the PTH(1-34) leads to conformational transitions characterized by decrease in FRET efficiency while NH(2)-terminal truncated ligand PTH(3-34) stimulates conformational transitions characterized by higher FRET efficiencies. 3) Stimulation of murine preosteoblastic cells (MC3T3-E1) with fluid shear stress (FSS) leads to significant changes in conformational equilibrium of the PTH1R in MC3T3-E1 cells, suggesting that mechanical perturbation of the plasma membrane leads to ligand-independent response of the PTH1R. Conformational transitions induced by mechanical stress were characterized by an increase in FRET efficiency, similar to those induced by the NH(2)-terminal truncated ligand PTH(3-34). The response to the FSS stimulation was inhibited in the presence of PTH(1-34) in the flow medium. These results indicate that the FSS can modulate the action of the PTH(1-34) ligand. 4) Plasma membrane fluidization using benzyl alcohol or cholesterol extraction also leads to conformational transitions characterized by increased FRET levels. We therefore suggest that PTH1R is involved in mediating primary mechanochemical signal transduction in MC3T3-E1 cells.

Molecular dynamics study of chemically engineered green fluorescent protein mutants: Comparison of intramolecular fluorescence resonance energy transfer rate

Because of its unusual spectroscopic properties, green fluorescent protein (GFP) has become a useful tool in molecular genetics, biochemistry and cell biology. Here, we computationally characterize the behavior of two GFP constructs, designed as bioprobes for enzymatic triggering using intramolecular fluorescence resonance energy transfer (FRET). These constructs differ in the location of an intramolecular FRET partner, an attached chemical chromophore (either near an N-terminal or C-terminal site). We apply the temperature replica exchange molecular dynamics method to the two flexible constructs in conjunction with a generalized Born implicit solvent model. The calculated rate of FRET was derived from the interchromophore distance, R, and orientational factor, kappa(2). In agreement with experiment, the construct with the C-terminally attached dye was predicted to have higher energy transfer rate than observed for the N-terminal construct. The molecular basis for this observation is discussed. In addition, we find that the orientational factor, kappa(2), deviates from the commonly assumed value, the implications of which are also considered.

Dendritic oligothiophene-perylene bisimide hybrids: synthesis, optical and electrochemical properties

Novel dendritic oligothiophene-perylene bisimide hybrid (DOTPBI) systems were investigated. Perylene bisimide (PBI) derivatives with phenoxy spacers bearing dendritic oligothiophenes (DOT) up to the third generation (G3) in the bay position, with an increasing number of conjugated thiophene units have been synthesized and characterized as novel functional materials. Investigation of the optoelectronic properties revealed that the perylene core and dendritic oligothiophene units are electronically decoupled. In the DOTPBIs intramolecular fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) from the DOT to the PBI moiety have been investigated by various optical measurements, revealing that the PET process is facilitated with increasing DOT generation and donor strength. Electropolymerization led to interesting cross-linked donor acceptor-type conducting polymer films.

Probing Protein Conformations by in Situ Non-Covalent Fluorescence Labeling

The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for site-specific incorporation of FRET acceptors into proteins, which were covalently labeled with a donor fluorophore. We demonstrate versatile application of this approach for exploring the conformation of the type I interferon receptor ectodomains ifnar1-EC and ifnar2-EC. Substantial ligand-induced conformational changes of ifnar1-EC, but not ifnar2-EC, were observed by monitoring the fluorescence intensity and the fluorescence lifetime of the FRET donor. Time-resolved fluorescence correlation spectroscopy revealed a substantial conformational flexibility of ifnar1-EC and a ligand-induced tightening. Our results demonstrate that protein labeling with tris-NTA/fluorophores enables for efficient quantitative intramolecular FRET analysis.

Calpain and Proteasomal Regulation of Antiretroviral Zinc Finger Protein OTK18 in Human Macrophages: Visualization in Live Cells by Intramolecular FRET

As part of the innate immune defense against HIV infection, OTK18, a zinc finger protein, is upregulated in human macrophages and reduces viral replication through suppression of viral long-terminal repeat promoter activity. Although we know that the processing products of OTK18 accumulate in the cytoplasm of brain perivascular macrophages in advanced HIV encephalitis cases, the molecular mechanisms behind its post-translational processing are still poorly understood. To characterize OTK18 processing, we assessed a panel of protease inhibitors to identify the candidates involved in the OTK18 processing using human monocyte-derived macrophages (MDM) overexpressing OTK18 by recombinant adenoviral gene transfer. Viral infection of MDM strongly increased the processing of OTK18 into its N-terminal fragment. Treatment of OTK18-expressing MDM with calpain and proteasome inhibitors significantly accumulated either full-length or processed OTK18 fragments in time- and dose-dependent manners. A series of OTK18 truncation mutants and synthetic peptides were tested to locate the calpain cleavage sites after arginine 359. Finally, we developed an enhanced cyan and yellow fluorescent protein (ECFP and EYFP)-based intramolecular fluorescent resonance energy transfer (intramolecular FRET) system to monitor the OTK18 endoproteolysis in human microglia cell line. Inhibition of proteasome activity significantly increased the intramolecular FRET signal in the nucleus. These data suggest that calpain and proteasome are involved in OTK18 endoproteolysis and degradation. Additionally, intramolecular FRET has proven to be a useful tool for monitoring the processing in live cells.

Fluorescence resonance energy transfer and anisotropy reveals both hetero- and homo-energy transfer in the pleckstrin homology-domain and the parathyroid hormone-receptor

We present a method and an apparatus of polarized fluorescence resonance energy transfer (FRET) and anisotropy imaging microscopy done in parallel for improved interpretation of the photophysical interactions. We demonstrate this apparatus to better determine the protein-protein interactions in the pleckstrin homology domain and the conformational changes in the Parathyroid Hormone Receptor, a G-protein coupled receptor, both fused to the cyan and yellow fluorescent proteins for either inter- or intramolecular FRET. In both cases, the expression levels of proteins and also background autofluorescence played a significant role in the depolarization values measured in association with FRET. The system has the sensitivity and low-noise capability of single-fluorophore detection. Using counting procedures from single-molecule methods, control experiments were performed to determine number densities of green fluorescence protein variants CFP and YFP where homo resonance energy transfer can occur. Depolarization values were also determined for flavins, a common molecule of cellular background autofluorescence. From the anisotropy measurements of donor and acceptor, the latter when directly excited or when excited by energy transfer, we find that our instrumentation and method also characterizes crucial effects from homotransfer, polarization specific photobleaching and background molecules.

Fluorescence-Enhanced Europium-Diethylenetriaminepentaacetic (DTPA)-Monoamide Complexes for the Assessment of Renal Function

Real-time, noninvasive assessment of glomerular filtration rate (GFR) is essential not only for monitoring critically ill patients at the bedside, but also for staging and monitoring patients with chronic kidney disease. In our pursuit to develop exogenous luminescent probes for dynamic optical monitoring of GFR, we have prepared and evaluated Eu(3+) complexes of several diethylenetriamine pentaacetate (DTPA)-monoamide ligands bearing molecular "antennae" to enhance metal fluorescence via intramolecular ligand-metal fluorescence resonance energy transfer process. The results show that Eu-DTPA-monoamide complex 18b, which contains a quinoxanlinyl antenna, exhibits large (ca. 2700-fold) Eu(3+) fluorescence enhancement. Indeed, complex 18b exhibits the highest fluorescent enhancement observed thus far in the DTPA-type metal complexes. The renal clearance property was assessed using the corresponding radioactive (111)In complex 18a, and the data suggest that this complex clears via a complex mechanism that includes glomerular filtration.

β2-Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca(2+) pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca(2+)-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45A. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca(2+)-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.

FRET-derived ratiometric fluorescence sensor for Cu 2+

New N-(pyrenylmethyl)naphtho-azacrown-5 ( 1) was synthesized as an ‘On–Off’ fluorescent chemosensor for Cu 2+. Excited at 240 nm corresponding to the absorption of naphthalene unit (energy donor) of 1, emission at 380 nm from pyrene unit (energy acceptor) is observed, indicating that intramolecular fluorescence resonance energy transfer (FRET-On) occurs in 1. When Cu 2+ is added to a solution of 1, however, the fluorescence of pyrene is strongly quenched (FRET-Off) whereas that of naphthalene group is revived. Such FRET ‘On–Off’ behavior of 1 is observed only in the case of Cu 2+ binding, but not for other metal cations. The high selectivity of 1 toward Cu 2+ can be potentially applied to a new kind of FRET-based chemosensor. The FRET On–Off behavior is supported by computational studies. The calculated molecular orbitals of HOMO and LUMOs suggest the excited-state interactions leading to FRET from naphthalene to pyrene in 1, but no electron density changes in 1·Cu 2+ complex.

Ternary complex formation of pVHL, elongin B and elongin C visualized in living cells by a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique

The tumor suppressor von Hippel-Lindau (VHL) gene product forms a complex with elongin B and elongin C, and acts as a recognition subunit of a ubiquitin E3 ligase. Interactions between components in the complex were investigated in living cells by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM). Elongin B-cerulean or cerulean-elongin B was coexpressed with elongin C-citrine or citrine-elongin C in CHO-K1 cells. FRET signals were examined by measuring a change in the fluorescence lifetime of donors and by monitoring a corresponding fluorescence rise of acceptors. Clear FRET signals between elongin B and elongin C were observed in all combinations, except for the combination of elongin B-cerulean and citrine-elongin C. Although similar experiments to examine interaction between pVHL30 and elongin C linked to cerulean or citrine were performed, FRET signals were rarely observed among all the combinations. However, the signal was greatly increased by coexpression of elongin B. These results, together with results of coimmunoprecipitation experiment using pVHL, elongin C and elongin B, suggest that a conformational change of elongin C and/or pVHL was induced by binding of elongin B. The conformational change of elongin C was investigated by measuring changes in the intramolecular FRET signal of elongin C linked to cerulean and citrine at its N- and C-terminus, respectively. A strong FRET signal was observed in the absence of elongin B, and this signal was modestly increased by coexpression of elongin B, demonstrating that a conformation change of elongin C was induced by the binding of elongin B.

Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection

Liquid chromatography (LC) with fluorescence detection is described for simultaneous determination of native fluorescent bioamines (indoleamines and catecholamines). This is based on intramolecular fluorescence resonance energy transfer (FRET) in an LC system following post-column derivatization of native fluorescent bioamines’ amino groups with o-phthalaldehyde (OPA). OPA fluorescence was achieved through an intramolecular FRET process when the molecules were excited at maximum excitation wavelength of the native fluorescent bioamines. Bioamines separated by reversed-phase LC on ODS column were derivatized with OPA and 2-mercaptoethanol. This method provides sufficient selectivity and sensitivity for the determination of normetanephrine, dopamine, tyrosine, 5-hydroxytryptamine, tryptamine, and tryptophan in healthy human urine without prior sample purification.

Imaging FRET standards by steady‐state fluorescence and lifetime methods

Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.

Selective Determination of Tryptophan-containing Peptides through Precolumn Derivatization and Liquid Chromatography Using Intramolecular Fluorescence Resonance Energy Transfer Detection

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.

Differential mitotic degradation of the CDC25B phosphatase variants

CDC25 phosphatases control cell-cycle progression by dephosphorylating and activating cyclin-dependent kinases. CDC25B, one of the three members of this family in human cells, is thought to regulate initial mitotic events. CDC25B is an unstable protein whose proteasomal degradation is proposed to be controlled by beta-TrCP. Here, we have investigated the regulation of CDC25B during mitosis, using time-lapse video microscopy. We found that CDC25B expression is high during early mitosis, and that its degradation occurs after the metaphase-anaphase transition and cyclin B1 destruction. We also show that CDC25B degradation after metaphase is dependent on the integrity of the KEN-box and RRKSE motifs that are located within the alternatively spliced B domain, and that the CDC25B2 splice variant, that lacks this domain, is stable during mitosis. Furthermore, we show that the N-terminal region of CDC25B, encompassing the B domain, undergoes major conformational changes during mitosis that can be monitored by intramolecular fluorescence resonance energy transfer variation of specific CDC25B biosensors. This study demonstrates that CDC25B splice variants have differential mitotic stabilities, a feature that is likely to have major consequences on the local control of cyclin-dependent kinase-cyclin activities during mitotic progression.

Mapping the Architecture of Secretin Receptors with Intramolecular Fluorescence Resonance Energy Transfer Using Acousto-Optic Tunable Filter-Based Spectral Imaging

The molecular structure and agonist-induced conformational changes of class II G protein-coupled receptors are poorly understood. In this work, we developed and characterized a series of dual cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged secretin receptor constructs for use in various functional and fluorescence analyses of receptor structural variants. CFP insertions within the first or second intracellular loop domains of this receptor were tolerated poorly or partially, respectively, in receptors tagged with a carboxyl-terminal yellow fluorescent protein that itself had no effect on secretin binding or cAMP production. A similar CFP insertion into the third intracellular loop resulted in a plasma membrane-localized receptor that bound secretin and signaled normally. This fully active third-loop variant exhibited a significant decrease in fluorescence resonance energy transfer signals that were recorded with an acousto-optic tunable filter microscope after exposure to secretin agonist but not to a receptor antagonist. These data demonstrate changes in the relative positions of intracellular structures that support a model for secretin receptor activation.