Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.     

Ligand-induced conformational changes in the acetylcholine-binding protein analyzed by hydrogen-deuterium exchange mass spectrometry

Recent x-ray crystallographic studies of the acetylcholine-binding protein (AChBP) suggest that loop C, found at the circumference of the pentameric molecule, shows distinctive conformational changes upon antagonist and agonist occupation. We have employed hydrogen-deuterium exchange mass spectrometry to examine the influence of bound ligands on solvent exposure of AChBP. Quantitative measurements of deuterium incorporation are possible for approximately 56% of the Lymnaea AChBP sequence, covering primarily the outer surface of AChBP. In the apoprotein, two regions flanking the ligand occupation site at the subunit interface, loop C (residues 175-193) and loop F (residues 164-171), show greater extents of solvent exchange than other regions of the protein including the N- and C-terminal regions. Occupation by nicotinic agonists, epibatidine and lobeline, and nicotinic antagonists, methyllycaconitine, alpha-bungarotoxin, and alpha-cobratoxin, markedly restricts the exchange of loop C amide protons, influencing both the rates and degrees of exchange. Solvent exposure of loop C and its protection by ligand suggest that in the apoprotein, loop C exhibits rapid fluctuations in an open conformation. Bound agonists restrict solvent exposure through loop closure, whereas the larger antagonists restrict solvent exposure largely through occlusion of solvent. Loop F, found on the complementary subunit surface at the interface, also reveals ligand selective changes in amide proton exchange rates. Agonists do not affect solvent accessibility of loop F, whereas certain antagonists cause subtle accessibility changes. These results reveal dynamic states and fluctuating movements in the vicinity of the binding site for unligated AChBP that can be influenced selectively by ligands.     

Reversibly bound and covalently attached ligands induce conformational changes in the omega loop, Cys69-Cys96, of mouse acetylcholinesterase

We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase (mAChE). Six cysteine-substituted sites of mAChE (Leu(76), Glu(81), Glu(84), Tyr(124), Ala(262), and His(287)) were labeled with the environmentally sensitive fluorophore, acrylodan, and the kinetics of substrate hydrolysis and inhibitor association were examined along with spectroscopic characteristics of the acrylodan-conjugated, cysteine-substituted enzymes. Residue 262, being well removed from the active center, appears unaffected by inhibitor binding. Following the binding of ligand, hypsochromic shifts in emission of acrylodan at residues 124 and 287, located near the perimeter of the gorge, reflect the exclusion of solvent and a hydrophobic environment created by the associated ligand. By contrast, the bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (Omega loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent. The magnitude of fluorescence emission shift is largest at position 84 and smallest at position 76, indicating that a concerted movement of residues on the Omega loop accompanies gorge closure upon ligand binding. Acrylodan modification of substituted cysteine at position 84 reduces ligand binding and steady-state kinetic parameters between 1 and 2 orders of magnitude, but a similar substitution at position 81 only minimally alters the kinetics. Thus, combined kinetic and spectroscopic analyses provide strong evidence that conformational changes of the Omega loop accompany ligand binding.     

Photoaffinity labeling of the acetylcholine binding sites on the nicotinic receptor by an aryldiazonium derivative

p-(Dimethylamino)benzenediazonium fluoroborate (DDF) behaves, in the dark, as a reversible competitive antagonist of the electrical response of Electrophorus electricus electroplaque to acetylcholine and of the acetylcholine-gated single-channel currents recorded in the C2 mouse cell line. This chemically stable but highly photoreactive compound binds irreversibly to the acetylcholine receptor when irradiated by visible light. In vivo, it irreversibly blocks the postsynaptic response of E. electricus electroplaque to agonists. In vitro, it reduces the alpha-bungarotoxin-binding capacity of acetylcholine receptor rich membrane fragments prepared from Torpedo marmorata electric organ. Once reversibly bound to the T. marmorata acetylcholine receptor, this ligand can be selectively photodecomposed by an energy-transfer reaction involving a tryptophan residue(s) of the protein. By use of reagent concentrations that are below the dissociation constant at equilibrium, up to 60% of the agonist-binding sites are covalently labeled. Under these conditions the alpha subunit of the acetylcholine receptor is preferentially labeled, and this labeling is partially prevented by agonists or competitive antagonists. This protective effect is substantially increased by prior incubation with phencyclidine, a compound known to prevent the binding of DDF at the level of the high-affinity site for noncompetitive blockers [Kotzyba-Hibert, F., Langenbuch-Cachat, J., Jaganathen, J., Goeldner, M. P., & Hirth, C. G. (1985) FEBS Lett. 182, 297-301]. The incorporation of about one molecule of label in an agonist/competitive antagonist protectable manner per alpha-bungarotoxin-binding site suffices to fully block alpha-bungarotoxin binding to the membrane-bound receptor. Thus, DDF behaves as a monovalent photoaffinity label of the acetylcholine-binding site.     

A fluorescent alpha-factor analogue exhibits multiple steps on binding to its G protein coupled receptor in yeast

The yeast alpha-factor receptor encoded by the STE2 gene is a member of the extended family of G protein coupled receptors (GPCRs) involved in a wide variety of signal transduction pathways. We report here the use of a fluorescent alpha-factor analogue [K(7)(NBD), Nle(12)] alpha-factor (Lys(7) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), norleucine(12) alpha-factor) in conjunction with flow cytometry and fluorescence microscopy to study binding of ligand to the receptor. Internalization of the fluorescent ligand following receptor binding can be monitored by fluorescence microscopy. The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a sensitive and reproducible assay that can be conducted at low cell densities and is relatively insensitive to fluorescence of unbound and nonspecifically bound ligand. Using this assay, we determined that some receptor alleles expressed in cells lacking the G protein alpha subunit exhibit a higher equilibrium binding affinity for ligand than the same alleles expressed in isogenic cells containing the normal complement of G protein subunits. On the basis of time-dependent changes in the intensity and shape of the emission spectrum of [K(7)(NBD),Nle(12)] alpha-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment, followed by a conversion to a state in which the fluorophore moves to a more polar environment.     

Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations

Upon ligand binding at the subunit interfaces, the extracellular domain of the nicotinic acetylcholine receptor undergoes conformational changes, and agonist binding allosterically triggers opening of the ion channel. The soluble acetylcholine-binding protein (AChBP) from snail has been shown to be a structural and functional surrogate of the ligand-binding domain (LBD) of the receptor. Yet, individual AChBP species display disparate affinities for nicotinic ligands. The crystal structure of AChBP from Aplysia californica in the apo form reveals a more open loop C and distinctive positions for other surface loops, compared with previous structures. Analysis of Aplysia AChBP complexes with nicotinic ligands shows that loop C, which does not significantly change conformation upon binding of the antagonist, methyllycaconitine, further opens to accommodate the peptidic antagonist, alpha-conotoxin ImI, but wraps around the agonists lobeline and epibatidine. The structures also reveal extended and nonoverlapping interaction surfaces for the two antagonists, outside the binding loci for agonists. This comprehensive set of structures reflects a dynamic template for delineating further conformational changes of the LBD of the nicotinic receptor.     

Conformational variability of the glycine receptor M2 domain in response to activation by different agonists

Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric alpha1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19' or 22' positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19'C increased by approximately 20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and beta-alanine were weak partial agonists at the alpha1R19'C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or beta-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22'C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists.     

Spectroscopic and functional characterization of an environmentally sensitive fluorescent actin conjugate

Rabbit skeletal muscle F-actin has been selectively labeled at a cysteine residue with the environmentally sensitive fluorophore 6-acryloyl-2-(dimethylamino)naphthalene. The fluorescent actin conjugate behaves similarly to native actin with respect to the polymerization kinetics, critical monomer concentration, and ability to form F-actin paracrystals. Upon polymerization to F-actin, the absorption of the actin conjugate is red-shifted, whereas the fluorescence emission is blue-shifted 740 wavenumbers and is accompanied by a decrease in the fluorescence bandwidth of 470 wavenumbers. These large shifts in the spectral properties of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) in actin provide a simple method for obtaining a spectral discrimination between the G- and F-actin populations during the polymerization reaction. Steady-state fluorescence techniques were used to study the environment of the fluorophore in the monomeric and polymeric forms of actin. Fluorescence emission spectral analysis and quenching and polarization studies of G-actin-Prodan indicated that the fluorophore lies immobile on the protein surface but with one of its faces in full contact with the solvent. In F-actin, the fluorophore has a limited exposure to the solvent and is located in a dielectric environment similar to those seen for Prodan in polar, aprotic solvents or buried within a protein matrix [Macgregor, R. B., Jr., & Weber, G. (1986) Nature (London) 318, 70-73]. Additionally, our results demonstrate that the Prodan molecule conjugated to F-actin is completely immobile during its fluorescence lifetime, exhibits an increase in the resonance energy transfer (RET) from tryptophan residues compared to that observed in G-actin, and shows evidence of homologous RET within the polymer.     

Characterization of the binding domain of the beta-adrenergic receptor with the fluorescent antagonist carazolol. Evidence for a buried ligand binding site

The antagonist carazolol has been used as a fluorescent probe for the binding site of the beta-adrenergic receptor (beta AR). The fluorescence properties of carazolol are dominated by the emission of the carbazole group, with the fine structure of the spectrum, but not the quantum yield, sensitive to the environment of the probe. The fluorescence emission spectrum of the bound probe is consistent with an extremely hydrophobic environment in the binding site of the receptor. Binding of carazolol to the purified beta AR increases the polarization of the fluorophore. Exposure to collisional quenchers has demonstrated the bound carazolol to be completely inaccessible to the solvent. Furthermore, the fluorescence of bound carazolol is not quenched by exposure to sodium nitrite, a F?rster energy acceptor which has an R0 value of 11.7 A with carazolol. Thus, physical analysis of the binding site of the beta AR by carazolol fluorescence indicates that the antagonist binds to the beta AR in a rigid hydrophobic environment which is buried deep within the core of the protein.     

Agonist-induced conformational changes at the cytoplasmic side of transmembrane segment 6 in the beta 2 adrenergic receptor mapped by site-selective fluorescent labeling

The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of agonist-induced conformational changes in the beta(2) adrenergic receptor, a prototype hormone-activated G protein-coupled receptor. In the background of a mutant beta(2) adrenergic receptor, with a minimal number of endogenous cysteine residues, new cysteines were introduced in positions 269(6.31), 270(6.32), 271(6.33), and 272(6.34) at the cytoplasmic side of transmembrane segment (TM) 6. The resulting mutant receptors were fully functional and bound both agonists and antagonist with high affinities also upon IANBD labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants suggested that the covalently attached fluorophore was exposed to a less polar environment at all four positions upon agonist binding. Whereas evidence for only a minor change in the molecular environment was obtained for positions 269(6.31) and 270(6.32), the full agonist isoproterenol caused clear dose-dependent and reversible increases in fluorescence emission at positions 271(6.33) and 272(6.34). The data suggest that activation of G protein-coupled receptors, which are activated by "diffusible" ligands, involves a structural rearrangement corresponding to the cytoplasmic part of TM 6. The preferred conformations of the IANBD moiety attached to the inserted cysteines were predicted by employing a computational method that incorporated the complex hydrophobic/hydrophilic environment in which the cysteines reside. Based on these preferred conformations, it is suggested that the spectral changes reflect an agonist-promoted movement of the cytoplasmic part of TM 6 away from the receptor core and upwards toward the membrane bilayer.     

Competitive binding assay using fluorescence resonance energy transfer for the identification of calmodulin antagonists

The ubiquitous calcium regulating protein calmodulin (CaM) has been utilized as a model drug target in the design of a competitive binding fluorescence resonance energy transfer assay for pharmacological screening. The protein was labeled by covalently attaching the thiol-reactive fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to an engineered C-terminal cysteine residue. Binding of the environmentally sensitive hydrophobic probe 2,6-anilinonaphthalene sulfonate (2,6-ANS) to CaM could be monitored by an increase in the fluorescence emission intensity of the 2,6-ANS. Evidence of fluorescence resonance energy transfer (FRET) from 2,6-ANS (acting as a donor) to MDCC (the acceptor in this system) was also observed; fluorescence emission representative of MDCC could be seen after samples were excited at a wavelength specific for 2,6-ANS. The FRET signal was monitored as a function of the concentration of calmodulin antagonists in solution. Calibration curves for both a selection of small molecules and a series of peptides based upon known CaM-binding domains were obtained using this system. The assay demonstrated dose-dependent antagonism by analytes known to hinder the biological activity of CaM. These data indicate that the presence of molecules known to bind CaM interfere with the ability of FRET to occur, thus leading to a concentration-dependent decrease of the ratio of acceptor:donor fluorescence emission. This assay can serve as a general model for the development of other protein binding assays intended to screen for molecules with preferred binding activity.     

Crystal structure of a Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors

The crystal structure of the snake long alpha-neurotoxin, alpha-cobratoxin, bound to the pentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis, was solved from good quality density maps despite a 4.2 A overall resolution. The structure unambiguously reveals the positions and orientations of all five three-fingered toxin molecules inserted at the AChBP subunit interfaces and the conformational changes associated with toxin binding. AChBP loops C and F that border the ligand-binding pocket move markedly from their original positions to wrap around the tips of the toxin first and second fingers and part of its C-terminus, while rearrangements also occur in the toxin fingers. At the interface of the complex, major interactions involve aromatic and aliphatic side chains within the AChBP binding pocket and, at the buried tip of the toxin second finger, conserved Phe and Arg residues that partially mimic a bound agonist molecule. Hence this structure, in revealing a distinctive and unpredicted conformation of the toxin-bound AChBP molecule, provides a lead template resembling a resting state conformation of the nicotinic receptor and for understanding selectivity of curaremimetic alpha-neurotoxins for the various receptor species.     

Characterization of ligand-induced conformational states in the beta 2 adrenergic receptor

Drugs acting at G protein coupled receptors can be classified in biological assays as either agonists, partial agonists, neutral antagonists, or as inverse agonists. Very little is known about the actual molecular events and structural changes that occur in the receptor following ligand binding and during transmission of a signal across the membrane. Therefore, the structural basis for the biological classification of drug action remains unknown. To date, the conformational state of G protein coupled receptors has been inferred from the activity of the effector enzyme modulated by the G protein. We have used two different approaches to monitor conformational changes in beta 2 adrenergic receptor. Fluorescence spectroscopy can be used to directly monitor structural changes in purified beta 2 adrenergic receptor in real-time. The emission from many fluorescent molecules is strongly dependent on the polarity of the environment in which they are located. Thus, fluorescent probes covalently bound to proteins can be used as sensitive indicators of conformational changes and protein-protein interactions. In addition, we examined functional differences between agonists and partial agonists using fusion proteins between wild-type beta 2 receptor or a constitutively active beta 2 receptor mutant and Gs alpha. These receptor-G protein fusion proteins guarantee highly efficient coupling with a defined stoichiometry. The results of these experiments will be discussed in the context of current models of G protein coupled receptor activation.     

A new fluorescence complementation biosensor for detection of estrogenic compounds

Estrogenic compounds are an important class of hormonal substances that can be found as environmental contaminants, with sources including pharmaceuticals, human and animal waste, the chemical industry, and microbial metabolism. Here we report the creation of a biosensor useful for monitoring such compounds, based on complementation of fluorescent protein fragments. A series of sensors were made consisting of fragments of a split mVenus fluorescent protein fused at several different N-terminal and C-terminal positions flanking the ligand binding domain of the estrogen receptor alpha. When expressed in HeLa cells, sensor 6 (ERα 312-595) showed a nine-fold increase in fluorescence in the presence of estrogen receptor agonists or antagonists. Sensor 2 (ERα 281-549) discriminated between agonists and antagonists by showing a decrease in fluorescence in the presence of agonists while being induced by antagonists. The fluorescent signal of sensor 6 increased over a period of 24 h, with a two-fold induction visible at 4 h and four-fold at 8 h of ligand incubation. Ligand titration showed a good correlation with the known relative binding affinities of the compound. The sensor could detect a number of compounds of interest that can act as environmental endocrine disruptors. The lack of a substrate requirement, the speed of signal development, the potential for high throughput assays, and the ability to distinguish agonists from antagonists make this an attractive sensor for widespread use.     

Orientation of d-tubocurarine in the muscle nicotinic acetylcholine receptor-binding site

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist, d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR gamma-subunit. Because data for mutations at residue gammaTyr(117) were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of gammaTyr(117) with both the 2-N and the 13'-positions of dTC. Examination of a dTC analog with a negative charge at the 13'-position failed to reveal electrostatic interaction with charged side-chain substitutions at gamma117, but the effects of side-chain substitutions remained consistent with proximity of Tyr(117) to the cationic 2-N of dTC. The apparent interaction of gammaTyr(117) with the 13'-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-pi electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to gammaTyr(117). This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.     

Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 1. Equilibrium studies.

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica are described. One class of saturable ethidium sites was blocked by alpha-bungarotoxin and therefore reflects direct binding to the receptor (Kd approximately 3 micrometers; stoichiometry--one ethidium site per two alpha-bungarotoxin sites). The second class of sites was nonsaturable and unaffected by alpha-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the dissociation constant and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anaesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in flourescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium appears not to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.     

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.     

Protease-sensitive signalling by chemically engineered intramolecular fluorescent resonance energy transfer mutants of green fluorescent protein

The native cysteine residues of green fluorescent protein (GFP) at positions 48 and 70 were replaced by non-thiolic amino acids, and new cysteine sites were introduced at specific, surface positions. Based on molecular modeling of the GFP structure, the sites chosen for mutagenesis to Cys were glutamic acid at position 6 and isoleucine at position 229. These new, unique cysteine sites provided reactive thiol groups suitable for site-specific chemical modification by eosin-based fluorescence labels. The new constructs were designed to serve as the basis of proof of principle for fluorescence resonance energy transfer (FRET) using an enzyme-activated (trypsin) intervening sequence between native and chemically conjugated fluorophores. These eosin moieties provided chemical FRET partners for the native GFP chromophore. On excitation, these GFP-eosin constructs exhibited strong intramolecular FRET, with quenching of the native GFP (511 nm) fluorophore emission and emission around 540 nm, corresponding to eosin. GFP mutants engineered with trypsin-sensitive sequences close to the eosin site, so that on trypsinolysis FRET was destroyed, the emission wavelength switching from that of the chemical FRET partner back to that of the native GFP fluorophore, providing efficient, ratio-based detection. This protein engineering provides the basis for novel bioprobes for enzymatic triggering using intramolecular FRET between GFP and carefully sited chemical labels.