Competitive Binding Assay Using Fluorescence Resonance Energy

Bioconjugate Chem. 2005, 16, 1257−1263

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Competitive Binding Assay Using Fluorescence Resonance Energy Transfer for the Identification of Calmodulin Antagonists Bethel Sharma,† Sapna K. Deo,‡ Leonidas G. Bachas,‡ and Sylvia Daunert*,†,‡ Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40536-0082, and Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055. Received June 6, 2005; Revised Manuscript Received August 12, 2005

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-(1maleimidyl)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.

INTRODUCTION

Fluorescence-based technologies are of great importance in pharmacological screening assays as they are amenable to miniaturization, and provide high sensitivity and signal-to-noise ratio. Fluorescence resonance energy transfer (FRET) is a valuable tool for characterizing proximity relationships within and between biomolecules (1-11). Many FRET assays make use of variants of the green fluorescent protein (GFP), in both in vitro and in vivo applications. In particular, fusion proteins between a target protein and two spectrally overlapping GFP’s have been used in these types of assays (12). One limitation of GFP-based FRET assays is that fusion of one (or two) GFP molecules to a protein could affect the folding of the hybrid protein. Additionally, the bulkiness of GFP could place constraints on any associated ligandinduced conformational changes, thus hindering the use of this fluorescent protein as a signal generator for bioanalysis. Consequently, development of FRET labeling strategies that minimize interference in the target protein’s normal folding and binding activities is advantageous. To that end, this manuscript describes the use of a FRET donor-acceptor pair that requires only a single residue modification that is distant from the binding site of the target protein for site-specific covalent attachment of the acceptor. The efficiency of energy transfer in FRET is inversely related to the distance between the donor and acceptor * To whom correspondence should be addressed: 217 Chemistry-Physics Building, University of Kentucky, Lexington, KY 40506. Phone: 859-257-7060, Fax: 859-323-1069, E-mail: [email protected]. † Department of Pharmaceutical Sciences. ‡ Department of Chemistry.

fluorophores. Proximity (less than 100 Å) is important in achieving nonradiative transfer between the two fluorophores, even if there is a significant overlap of the donor emission and acceptor excitation wavelengths. Thus, FRET assays are useful as a type of “molecular gauge” of proximity relationships. This allows for great sensitivity in the detection of structural transitions or alterations (13). As the human proteome becomes elucidated, new therapeutic targets are progressively identified. To evaluate libraries of drug candidates against these targets, initial screening assays for potency, selectivity, and activity are necessary. Accordingly, new screening assays that are fast, simple, with no complex separation steps, inexpensive, and easily amenable to miniaturization are critical tools in the drug discovery process. In that regard, we have used calmodulin (CaM) as a model drug target to demonstrate the feasibility of a newly designed method for rapid assessment of binding affinities of drug candidates in a homogeneous format. CaM is a small calciummodulated protein found in all eukaryotes that binds to and activates its numerous target proteins (14-18). Herein, we report the development of a screening assay to evaluate protein-binding activity employing FRET between a fluorescent molecule in the assay solution and CaM site-specifically labeled with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). This assay is used to assess binding of a selection of antagonists including both drug molecules and peptides representative of CaM-binding domains, in the presence of the hydrophobic fluorescent probe, 2,6-anilinonaphthalene sulfonate (2,6-ANS). Upon binding of 2,6-ANS to CaM, energy transfer occurs between 2,6-ANS, which binds to CaM but can be displaced by the analyte, and

10.1021/bc050161y CCC: $30.25 © 2005 American Chemical Society Published on Web 09/02/2005

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the covalently attached MDCC at the C-terminus of the protein. When there is drug or peptide present in the sample, the binding event displaces the noncovalently bound 2,6-ANS in a competitive manner. This consequently increases the distance between the two fluorophores, leading to a decrease in the observed emission signal from the FRET acceptor, MDCC. A change in the concentration of analyte present in the sample thereby alters the ratio of FRET acceptor to FRET donor fluorescence emission. We have constructed calibration curves to evaluate the response of a series of drugs and peptides with known affinity for calmodulin as well as selected control compounds. MATERIALS AND METHODS

Reagents. All test compounds, buffers, salts, the myosin light chain kinase inhibitor peptide, and CaMdependent protein kinase II-delta fragment peptide were purchased from Sigma (St. Louis, MO). All other CaM binding peptides were obtained from Calbiochem (San Diego, CA). Dithiothreitol (DTT) was acquired from Gold Biotech (St. Louis, MO). Isopropyl-β-D-thiogalactopyranoside (IPTG) and ampicillin were obtained from U.S. Biochemical (Cleveland, OH). Synthetic oligonucleotides used as primer sequences for PCR were prepared by Operon (Alameda, CA). Taq DNA polymerase, the protein expression kit, and any necessary restriction enzymes were purchased from New England Biolabs (Beverly, MA). The fluorescent probes MDCC and 2,6-ANS were obtained from Molecular Probes (Eugene, OR). The BioRad protein assay reagent was purchased from Bio-Rad Laboratories (Hercules, CA). Apparatus. Starter cell cultures were grown in an orbital shaker from Forma Scientific (Marietta, OH). Fermentation of bacterial cultures was performed using a bioreactor from New Brunswick Scientific (New Brunswick, NJ). Optical density of cell cultures was measured on a Spectronic 21-D spectrophotometer from Milton Roy (Rochester, NY). Protein samples were concentrated using a Christ lyophilizer (Osterode, Germany). Protein purity was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a PhastSystem from Pharmacia Biotech (Uppsala, Sweden). Fluorescence studies were carried out on either a Fluorolog-2 spectrofluorometer from Spex-JY Horiba (Edison, NJ) or a Cary Eclipse spectrofluorometer equipped with a multiwell plate reader from Varian (Palo Alto, CA). Protein Expression, Purification, and Labeling. Cloning. The New England Biolabs IMPACT-TWIN system was used to create a C-terminal fusion between a cleavable intein (solely for affinity purification on a chitin bead column) and a mutant CaM-containing a C-terminal cysteine residue. To generate the C-terminal cysteine CaM, a single round of PCR was performed with Taq DNA polymerase using the plasmid pVUC-1 (19) as a template. To add NdeI and SapI sites at the 5′- and 3′-termini, respectively, and include an insertion of a single cysteine at the C-terminus, the forward primer (CaMNdeI-F) 5′-GGTGGTGGTGGTGGCATATGATGGCTGATCAGCTGACT-3′ and the reverse primer (CaMSapI-R) 5′-GGTGGTGGTGGTTGCTCTTCCGCAACATGATCCT-CCTCCGGACTTAGCCATCATAACCTG-3′ were designed. Gene amplification was carried out for 30 cycles using a Perkin-Elmer Gene Amp thermal cycler (Norwalk, CT). The PCR denaturation was at 94 °C for 0.5 min, the annealing at 55 °C for 1 min, and the elongation at 72 °C for 1 min. The SapI and NdeI restriction sites were used to insert the PCR-amplified synthetic cal-

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modulin gene into the pTWIN1 vector. The plasmid containing the inserted gene was transformed into E. coli strain ER2566. Transformants were grown on selective media, and positive transformants were sequenced to verify the presence of the plasmid pTWIN1-CaM, containing the C-terminal cysteine CaM gene. Expression and Purification. The IMPACT-TWIN system was also used to express and purify the recombinant calmodulin. E. coli ER2566 cells harboring the pTWIN1CaM plasmid were grown at 37 °C in Luria Bertani medium containing 100 µg/mL ampicillin for approximately 16 h, until the OD600 reached 0.6. Protein expression was induced with a final concentration of 1 mM IPTG for 4 h followed by centrifugation for 20 min at 3800g to harvest the cells. The pellet was resuspended in a pH 7.0 buffer containing 20 mM Tris-HCl, 500 mM NaCl, and 1 mM EGTA. Following sonication to lyse the cells, and centrifugation to clarify the crude cell extract, the supernatant was loaded onto a 5-mL column of chitin beads equilibrated in the same 20 mM Tris-HCl buffer in order to selectively purify the CaM-chitin binding domain fusion protein from the cellular milieu. Two washes were performed to remove any remaining unbound protein. The first was with the same buffer used to resuspend the cells, and a second wash used the same buffer, but with the NaCl concentration increased to 1 M. Then, three column volumes of 20 mM Tris-HCl, pH 8.5, containing 500 mM NaCl and 40 mM DTT were passed through the column, the column flow was stopped, and the column was stored at 4 °C for 16 h while the on-column cleavage reaction occurred. The next day, three more column volumes of the DTT-containing buffer were used to elute the bound protein, releasing the CaM from the bound chitin-binding domain at the intein cleavage site. The mutant CaM was isolated at greater than 90% purity based on SDSPAGE. Protein concentrations were measured using the Bio-Rad protein reagent, a modified Bradford assay reagent. Further purification occurred after the labeling reaction. Labeling. The protein was labeled by covalently attaching the thiol-reactive fluorophore, MDCC to the engineered C-terminal cysteine residue on CaM. The concentration of the unlabeled protein fraction varied from batch to batch, but the molar ratio of fluorophore to protein in the reaction mixture was always approximately 10:1. The labeling reaction and subsequent separation of labeled protein from unbound fluorophore using size-exclusion chromatography was performed as in ref 29. The extent of MDCC labeling was assessed using the following equation:

Ax MWprotein moles dye ) × moles protein  × b mgprotein/mL where Ax is the absorbance at 420 nm (MDCC maximum),  is the extinction coefficient for MDCC ) 50 200 cm-1 M-1, and b is the path length in cm. Labeling efficiencies were typically around 70%. Fluorescence Studies of Interaction between Calmodulin and 2,6-ANS. Energy Transfer Studies. 2,6ANS binding to unlabeled CaM could be monitored by the increase in fluorescence emission intensity of the 2,6ANS when samples were excited at 360 nm and emission was measured at 420 nm. To observe energy transfer from 2,6-ANS to a 0.5 µM concentration of MDCC-labeled CaM, the donor excitation wavelength used was 360 nm, and the acceptor emission was measured at 460 nm using

FRET Assay for Calmodulin Antagonists

Figure 1. Crystal structure of Ca2+-CaM (1EXR) bound to the myosin light chain kinase CaM-binding domain peptide, shown in blue (1CDL), and to the antipsychotic drug trifluoperazine, shown in red (1CTR). Calcium ions are shown in yellow. Protein databank (PDB) (46) files are indicated in parentheses.

a conventional benchtop fluorometer and poly(methyl methacrylate) cuvettes. Both studies were performed in a buffer containing 20 mM Tris-HCl, pH 8.0, with calcium-saturated CaM-MDCC. Competitive Binding Experiments. CaM-MDCC was diluted to a final concentration of 0.5 µM in an assay volume of either 1.5 mL or 250 µL depending upon the assay platform used (conventional fluorometer or microtiter plate reader, respectively). The CaM solution was prepared in 20 mM Tris-HCl, pH 7.5, containing 100 mM KCl, 10 mM CaCl2, and 0.5 µM 2,6-ANS. Each test sample was diluted to the final concentration with either buffer alone or varying concentrations of drug prepared in the buffer solution. The fluorescence was measured directly after a brief mixing step. Samples were excited at the donor excitation wavelength of 360 nm (2,6-ANS), and donor and acceptor emissions were measured at 410 nm (2,6-ANS) and 460 nm (MDCC). The ratio of acceptor to donor fluorescence was plotted versus analyte concentration to construct calibration curves. RESULTS AND DISCUSSION

The crystal structures of three different conformations of calmodulin (Ca2+-CaM, Ca2+-peptide-CaM, and Ca2+TFP-CaM) are displayed in Figure 1. On the right, trifluoperazine (TFP) is shown bound to the C-terminal domain of the globular protein. While it is commonly acknowledged that the CaM-trifluoperazine complex has a 1:1 stoichiometry, evidence has been offered (including crystallographic data) that CaM can bind up to four molecules of trifluoperazine (TFP) in solution and form a compact globular structure (20-23). Assays have been developed to measure interactions of CaM with drugs and biomolecules previously. Calmodulin has been employed since the early 1990s as a calcium sensor (24, 25). These biosensing systems utilized CaM labeled with fluorescent probes, either randomly or site selectively. In addition,

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other systems have been employed in ex vivo or in vivo Ca2+ detection utilizing the fusion of CaM to two flanking GFPs (5, 15, 16, 26-28). Also, two different systems for the detection of phenothiazine-type drugs for highthroughput screening applications using calmodulin have been presented (29, 30). Moreover, in another study a doubly labeled, agarose immobilized, CaM-maltose binding protein fusion protein was constructed for use in a FRET assay for single molecule detection of a CaMbinding peptide (31). It is well established that solvent-exposed hydrophobic surfaces are formed upon Ca2+ binding to CaM (14-16). Anilinonaphthalene sulfonates (ANS) can serve as fluorescent probes of hydrophobicity as their quantum yield and emission maxima are dependent upon their environment. In water, ANS is virtually nonfluorescent, but in nonpolar environments it emits a bright blue fluorescence with a quantum yield ∼0.70 (32). ANS has been used previously to measure the surface hydrophobic character of proteins and membranes, particularly during the folding/unfolding of proteins (33-36). LaPorte et al. demonstrated through differential binding in the presence and absence of calcium that there are 2.1 ANS binding sites in CaM (37). Tsurata and Sano later argued that under LaPorte’s experimental conditions, CaM could not be saturated with calcium, and that there were likely two to four ANS binding sites with binding constants ranging from 2.91 to 3.86 × 10-4 M-1 (38). Some investigators propose that the mode of ANS binding occurs through ion-pair formation between the sulfonate moiety and cationic groups on the protein (3941). In fact, it has been suggested that ANS may be a conformational tightening agent, and that ANS fluorescence may not be entirely due to interactions with hydrophobic sites on the protein, but instead to less interaction with water (40). Taking into consideration the discussion over the true character of ANS binding, we have developed a system that operates at a constant ANS concentration to overcome any possible discrepancies due to the number of ANS molecules bound at any given time to CaM. We have also set the ANS concentration at a level below that at which the inner filter effect causes the ANS fluorescence to decrease. In the presence of ANS and MDCC-labeled CaM, fluorescence was observed at the wavelength corresponding to the emission maximum of MDCC, with no direct excitation of the latter. This provided evidence that FRET could be monitored using our system. We achieved this by selectively exciting the ANS molecules, which exhibit weak fluorescence in aqueous solution and become noticeably fluorescent when CaM is present. We hypothesized that if ANS binds to CaM in its C-terminal hydrophobic region, it will be in close enough proximity to the C-terminal MDCC on the protein for fluorescence resonance energy transfer to occur, provided that the two molecules have sufficient spectral overlap. In fact, these two molecules were chosen because the emission of ANS and the excitation of MDCC are both close to 420 nm. Figure 2 depicts the principle of the FRET application described herein. On the right, a molecule of 2,6-ANS is shown near the hydrophobic binding pocket of the Cterminal domain of CaM, a potential binding site for ANS. In this instance, the bound MDCC at the Cterminus is in close enough proximity to the molecule of bound 2,6-ANS for energy transfer to occur. In the presence of phenothiazine-type compounds or CaMbinding peptides, a competitive binding interaction is introduced as the analyte (TFP in this case) replaces 2,6ANS in the hydrophobic pocket of CaM. By removing the

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Figure 2. Proposed scenario demonstrating the predicted energy transfer from the bound 2,6-ANS to the C-terminal attached MDCC. It is hypothesized that energy transfer is inhibited when the protein binds to drugs or model peptide molecules. The C-terminal residue, where MDCC is attached, is highlighted in orange and 2,6-ANS is black.

Figure 3. Excitation (- - -) and emission (___) spectra of 1 µM CaM-MDCC. The emission wavelength for the excitation trace was 460 nm. The excitation wavelength was set at 420 nm for the emission spectrum.

Figure 5. Effect of calcium concentration on energy transfer from 1 µM 2,6-ANS to 1 µM CaM-MDCC. 10 mM Ca2+ (- - -), 1 mM Ca2+ (___), and 1 mM EDTA (- - -) are shown. Excitation was at 360 nm.

Figure 4. Effect of changing calcium concentrations on 1 µM CaM-MDCC fluorescence. 10 mM Ca2+ (- - -), 1 mM Ca2+ (___), and 1 mM EDTA (- - -) are shown. Excitation was at 420 nm.

Figure 6. Effect of changing calcium concentrations on 0.25 µM CaM-MDCC with 1 µM 2,6-ANS. 10 mM Ca2+ (- - -), 1 mM Ca2+ (;), and 1 mM EDTA (___) are shown. The excitation was at 360 nm.

bound 2,6-ANS from CaM, FRET is limited as the 2,6ANS is too far away for any significant radiationless energy transfer to the bound MDCC molecule to occur. The excitation and emission spectra of 1 µM CaMMDCC in the presence of saturating calcium are shown in Figure 3. The effect of changing calcium concentrations on CaM-MDCC fluorescence was also assessed. In the presence of the metal complexing agent EDTA, the fluorescence of CaM-MDCC was lower than when Ca2+ was present (Figure 4). A concentration of 10 mM Ca2+ was

required to obtain maximum fluorescence emission at 460 nm. Thus, all assay buffers contained 10 mM calcium. We also considered the effect of calcium on the efficiency of the FRET interaction. The calcium dependence of FRET between the two chromophores using 1 µM 2,6ANS and 1 µM CaM-MDCC was studied, and the results are shown in Figure 5. The emission intensity at 460 nm was slightly higher when 10 mM calcium was present. Notice also that the ANS emission peak is shifted to 410 nm. The FRET ratios were thus calculated using 460/ 410 nm for the subsequent calibrations.

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FRET Assay for Calmodulin Antagonists Table 1. Description of Peptides Used in This Study synthetic peptide

amino acids

CaM inhibitory peptide CaM inhibitory peptide (control) CaM binding domain CaM kinase II inhibitor myosin light chain kinase inhibitor CaM dependent PK II-δ fragment(residues 345-358)

17 17

Ac-RRKWQKTGHAVRAIGRL-NH2 Ac-RRKEQKTGHAVRAIGRE-NH2

sequence

20 29 11

LKKFNARRKLKGAILTTMLA 2274.3 CaM kinase II residues 290-309 MHRQETVDCLKKFNARRKLKGAILTTMLA 3374.1 CaM kinase II residues 281-309 KRRWKKNFIAV-NH2 1445 chicken smMLCK CaM binding domain

14

KSDGGVKKRKSSS

CaM-MDCC could be detected at concentrations above 10 nM (shown in Supporting Information). We selected 0.25 µM as the final concentration for the assay to minimize the amount of protein used that could give a signal at least 100-fold higher than the blank when using the Varian Cary Eclipse microtiter plate reader. The appropriate concentration of 2,6-ANS to use in the assay was determined by monitoring the change in ANS fluorescence in the presence of the previously selected 0.25 µM CaM-MDCC concentration. The fluorescence of ANS quickly diminishes as the concentration of the molecule in solution increases due to the inner filter effect. Therefore, an assay concentration of 1 µM was determined to be appropriate for our system. We then combined all of these conditions and measured the emission in smaller volume (250 µL) microtiter plate samples with 0.25 µM CaM-MDCC and 1 µM 2,6-ANS and evaluated the effect of calcium on the energy transfer under the final assay conditions. A calcium concentration of 1 mM resulted in a higher emission signal than when EDTA was present. Figure 6 demonstrates that in this case, changes in calci-

mw

description

2074.5 based upon CaM binding domain of MLCK 2034.3 control for CaM inhibitory peptide

1450.6 substrate for brain Ca2+/CaM-dependent PK IV

um concentration from 1 mM to 10 mM did not visibly alter the acceptor (MDCC) fluorescence emission at 460 nm. Using the previously described conditions, energy transfer from 2,6-ANS to MDCC was observed and the ratio of acceptor to donor fluorescence intensity was related to the amount of drug or peptide present in each sample. Calibration curves for a series of peptides and drug compounds were obtained using this system. Table 1 and Figure 7 show the structures of all of the compounds that were tested using this assay, including negative controls that were used for selectivity testing (aspirin, propranolol, serotonin, and warfarin). The data indicate that the presence of known CaM antagonists interferes with the FRET activity, thus leading to a concentration-dependent decrease in the resultant fluorescence emission ratio. The observed changes could be a combination of dual effects, including the proposed displacement of 2,6-ANS, but also alteration of the local environment of the MDCC molecule upon drug or peptide binding. The graph in Figure 8 shows dose-response curves for changing concentrations of several peptides in the presence of CaM-MDCC and 2,6-ANS. Similar

Figure 8. Calibration plots for MLCK inhibitor peptide (b), CaM inhibitory peptide (2), CaM binding domain peptide (4), and CaM kinase II inhibitor peptide (9), are all described in Table 1.

Figure 7. Structures of test compounds. Compounds listed are known CaM antagonists and are classified as phenothiazine (chlorpromazine, thioridazine, and trifluoperazine), dibenzazepine (clozapine, haloperidol), or tricyclic antidepressant (imipramine) drugs. The other compounds listed are controls.

Figure 9. Calibration plots for the phenothiazine drugs chlorpromazine ([), thioridazine (2), and clozapine (O).

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to CaM. For the peptides in Figure 8, we ranked the percent inhibition of FRET relative to the FRET inhibition obtained with the CaM inhibitory peptide (Table 2). The Ca2+/CaM kinase II inhibitor peptide and CaM binding domain peptide, both derived from the CaM Kinase II CaM binding site, were found to be moderate FRET inhibitors, but had lower IC50 values than the CaM inhibitory peptide. A possible explanation for this difference in percent inhibition may relate to displacement of more than one molecule of 2,6-ANS per CaM. The myosin light chain kinase inhibitor peptide is a truncated version of the chicken smooth muscle myosin light chain kinase CaM binding domain. Thus, it was not surprising that is was the poorest inhibitor of FRET in our assay. CONCLUSION Figure 10. Calibration plots for CaM inhibitory peptide (control) (2), and CaM dependent PK II-δ fragment (0) demonstrate results similar to when no peptide is present (b). Table 2. Calculated IC50 Values Based upon Curve Fitting of Sigmoidal Dose-Response Curves Such as Those in Figures 8 and 9 peptide

IC50 (µM)

% FRET inhibition

CaM inhibitory peptide CaM kinase II inhibitor CaM binding domain MLCK inhibitor

0.731 0.204 0.232 1.424

100 57 45 12

a

drug

IC50 (µM)

known Ki (µM)a

imipramine thioridazine clozapine chlorpromazine trifluoperazine haloperidol

12.5 2.16 8.83 4.31 1.14 14.8

14 1.5 1.1 -

Data from Reynolds and Claxton (1982).

results with a few representative drug compounds can be seen in Figure 9. Results from all peptides and drugs tested that affected the FRET have been compiled and presented in Table 2. The IC50 values represent the effective concentration corresponding to 50% of the maximum acceptor:donor FRET ratio. It should be noted that no significant change in the FRET emission ratio was observed in the presence of compounds not known to affect the physiological behavior of CaM, such as aspirin and propranolol, which were used as negative controls (data not shown). Reynolds and Claxton have determined apparent Ki values for CaM with trifluoperazine, chlorpromazine, and imipramine, which are also listed in Table 2 (42). Their experiments measured the inhibition of CaM-activated guinea-pig heart phosphodiesterase by these compounds. IC50 values relate to the affinity of the interaction between ligands and receptors and have been shown by theoretical modeling to correlate with the KD values (43). The present study and that of Reynolds and Claxton are in agreement with respect to the order of affinity of calmodulin for certain drugs, namely, trifluoperazine > chlorpromazine > imipramine. The results from the peptides are indicative of their relative potencies. The CaM inhibitory peptide differs from the CaM inhibitory peptide (control) by only two residues selected to disrupt the interaction with calmodulin (44, 45). The control peptide showed no change in the FRET emission ratio at any concentration tested, and similar results were obtained with the Ca2+/CaM dependent protein kinase II-δ fragment as seen in Figure 10. The latter is actually a substrate for Ca2+/CaM dependent protein kinase IV and was not expected to bind

In summary, we have demonstrated the use of a competitive binding assay using FRET for the detection of drug and peptide antagonists of CaM. ANS has been shown to bind to hydrophobic binding sites and monitor binding events in a variety of proteins (34, 35}. ANS has also been used as a monitor of conformation and an indicator of protein folding (36, 37). Therefore, it is reasonable to conclude that ANS could be employed for applications in monitoring ligand-induced conformational changes in a wide range of binding proteins. Although ANS and the antagonists presumably bind at the same site in CaM, this is not a requirement for the type of FRET assay described herein. The only criterion is that the binding of a ligand to a protein binding site alters the binding constant of ANS to the protein. The two sites where ANS and the ligand bind need not be identical, and allosteric interaction would be sufficient. Thus, this FRET assay could be a model for other protein binding assays intended for use in pharmacological screening to detect compounds with desired properties. The assay has been proven to be rapid, simple, and economical, as it requires single rather than dual labeling for FRET. These are characteristics key to designing assays for drug candidate selection. The assay protocol developed also renders itself to automation, as it does not require complex separation steps. Finally, the sensitivity for both drug molecules and peptides has been demonstrated and compared among structurally related and unrelated compounds. ACKNOWLEDGMENT

This work was funded by the National Aeronautics and Space Administration and the National Science Foundation. B.S. gratefully recognizes support from an Integrated Graduate Education and Research Traineeship predoctoral fellowship. Supporting Information Available: CaM-MDCC calibration plot. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Szollosi, J., Nagy, P., Sebestyen, Z., Damjanovicha, S., Park, J. W., and Matyus, L. (2002) Applications of fluorescence resonance energy transfer for mapping biological membranes. J. Biotechnol. 82, 251-266. (2) Boute, N., Jockers, R., and Issad, T. (2002) The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol. Sci. 23, 351-354. (3) Heyduk, T. (2002) Measuring protein conformational changes by FRET/LRET. Curr. Opin. Biotechnol. 13, 292-296. (4) Kohl, T., Heinze, K. G., Kuhlemann, R., Koltermann A., and Schwille, P. (2002) A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc. Natl. Acad. Sci. U.S.A. 99, 12161-12166. (5) Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura M., and Tsien, R. Y. (1997) Fluorescent indicators

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