A Fluorescence Resonance Energy Transfer Sensor Based on

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Bioconjugate Chem. 2003, 14, 909−918

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A Fluorescence Resonance Energy Transfer Sensor Based on Maltose Binding Protein Igor L. Medintz,* Ellen R. Goldman, Michael E. Lassman, and J. Matthew Mauro*,† Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington D.C. 20375. Received December 3, 2002; Revised Manuscript Received April 2, 2003

A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E. coli maltose binding protein (MBP) is demonstrated. The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5). The novel acceptor portion of the sensor consists of β-cyclodextrin (β-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9. Binding of the modified β-CD to dye-conjugated MBP results in assembly of the FRET complex. Added maltose displaces the β-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety. In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 µM). Maltose limits of detection were in the 50-100 nm range.

INTRODUCTION

Although a variety of sensing approaches are available for sensitive real-time chemical detection, receptor-based or binding-protein-based biosensor development is a promising area (1-3). Incorporating a fluorescence resonance energy-transfer (FRET) reporter into a biosensor is especially appealing because of the potential sensitivity and specificity that can be achieved (4-7). Since FRET is intrinsically dependent upon and highly sensitive to proximity changes, it is an appealing monitoring/reporting mechanism for protein and molecular binding events and has been used extensively as a molecular or nanoscale spectroscopic ruler (4-7). FRET is defined as the nonradiative transfer of the excited-state energy from an initially excited donor to an acceptor (5, 6). Donor molecules emit at a shorter wavelength that corresponds to or overlaps a significant portion of the acceptor molecules absorption spectrum. The energy transfer efficiency, E, is defined as the number of quanta transferred to the acceptor divided by the number of quanta absorbed by the donor and is expressed as:

E)

1 1 + (R/R0)6

(1)

where R is the distance between the donor-acceptor pair and R0 is the donor-acceptor separation at 50% transfer efficiency (5, 6). Since there is a R-6 dependence, any change in distance between the donor-acceptor pair should translate to a dramatic change in energy transfer and hence fluorescence (5, 6). FRET has been the basis for many bioanalytical assays. For example, a Cy5-labeled antibody that bound to Cy5.5-labeled bovine serum albumin was used as a model for characterizing this particular FRET pair. This * Corresponding authors. M. Mauro: E-mail: matt.mauro@ probes.com. Tel: 591-465-8300. I. Medintz: E-mail: Imedintz@ cbmse.nrl.navy.mil. Tel: 202-404-6046. † Current address: Molecular Probes, Inc., 29851 Willow Creek Rd., Eugene, OR 97602.

Cy5-Cy5.5 FRET pair was subsequently applied to a homogeneous competitive immunoassay for the pesticide simazine and was shown to have a 0.3 µg/L lower limit of detection (7). FRET has also been widely used to study many protein-protein, protein-DNA, molecular structure, and other interactions (8, 9). The use of FRET in carbohydrate analysis has not been fully exploited and remains an exciting area of research (4, 10). An energy transfer sensor complex for glucose has been described (8). The donor consisted of concanavalin A labeled with a ruthenium metal-ligand complex bound, via maltose, to insulin labeled with malachite green. The assay functioned by glucose displacement of the maltose-insulin which increased the emission intensity and lifetime of the excited ruthenium-concanavalin A complex (8). Escherichia coli maltose binding protein (MBP) has proven to be an excellent model for prototyping and characterizing biosensing (3, 11-13). This periplasmic protein is involved in transcellular membrane transport of maltose and has been used in reagentless sensing of sugars. In early experiments, MBP methionine side chains were derivatized with the fluorescent reporter N-iodoacetyl-N′-(5-sulfo-1-naphythyl)ethylenediamine, IAEDANS (12). A systematic increase in FRET between a tryptophan residue of MBP and IAEDANS was detected upon addition of increasing concentrations of maltose, indicating that the MBP-IAEDANS construct functioned as a quantitative FRET biosensor (12). Other MBP-based sensors exploit the effects of allosteric changes in dyelabeled MBP structure upon maltose binding, which directly affects the intrinsic fluorescence intensity, allowing this sensor to function in a reagentless manner (3, 11). Allosterically based MBP sensor variants include fluorescently labeled mutants (11, 13), an electrochemically sensitive redox MBP conjugate (14), and an amperometric trinitrotoluene (TNT) sensing nitroreductaseMBP fusion protein (15). In fact, MBP has been proposed as a model for designing and prototyping allosterically active biosensor proteins (11). An interesting FRET-based MBP sensor has recently been described in which a MBP mutant was constructed with an enhanced cyan fluorescent protein (ECFP) at the N-terminus and an enhanced

10.1021/bc020062+ CCC: $25.00 © 2003 American Chemical Society Published on Web 08/30/2003

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Figure 1. Schematic representation of the FRET-based sensing scheme. The FRET complex (A) consists of Cy3.5-labeled MBP (donor) with bound β-CD-Cy5 (acceptor), resulting in FRET. Upon addition of maltose (B), the β-CD-Cy5 is displaced, resulting in increased MBP-Cy3.5 donor emission. If MBP-Cy3 and β-CD-QSY9 (a dark quencher) are substituted, the assembled complex results in quenching of the MBP-Cy3 donor emission.

yellow fluorescent protein (EYFP) at its C-terminus (16). Upon maltose binding, the dye moieties are brought into closer proximity which alters the FRET properties. This system functioned both in single yeast cells and in vitro (16). MBP binds a variety of sugars, with varying affinities including, β-cyclodextrin (β-CD). β-CD has been modified and incorporated in a variety of fluorescent sensing assays. These include a complexed boronic acid fluorophore-β-CD probe that selectively recognized sugars and transduced signal based on changes in fluorescence (17). Dansylglycine-modified β-CD immobilized on a cellulose membrane decreases its fluorescent intensity upon binding guest molecules (18). Dye-modified β-CD complexed to copper(II) was fluorescently quenched but ‘switched on’ fluorescence upon binding with amino acids (19). β-CD has also been conjugated to a peptide in close proximity to a pyrene moiety with guest interactions monitored fluorescently (20). Dextran labeled with malachite green has also been used in a FRET sensor for serum glucose (21). However, dye-modified β-CD has not been used directly in a FRET-based assay. In the work described in this report, we conjugated MBP with either of the long wavelength-excitable cyanine dyes Cy3 or Cy3.5. We also modified β-CD with the cyanine dye Cy5 or the dark quencher QSY9 and used the dye-modified protein and β-CD adduct as a FRETbased homogeneous biosensor. In this novel sensor, FRET is used to measure the concentration of maltose in solution by monitoring changes in fluorescence intensity of the donor moiety (see Figure 1 for a schematic). MBPCy3.5 was prepared at two different dye-to-protein ratios, 1 and 1.7, and then complexed with the β-CD-Cy5. MBP-Cy3 was also complexed with β-CD-QSY9 to enable formation of other homogeneous maltose sensor variants. One MBP-Cy3 variant was based upon MBP lysine residues modified by a Cy3-amine-active donor dye and another variant employed an engineered cysteine residue specifically labeled with Cy3-thiol-reactive energy donor dye. MATERIALS AND METHODS

Maltose Binding Protein. The DNA coding sequence for the MBP protein was contained on a standard multicopy plasmid vector containing the ampicillin resistance gene as described (11, 22). The MBP gene sequence was engineered to express a C-terminal fivehistidine sequence using standard gene assembly and cloning techniques (11, 22). A mutant MBP protein with aspartate-95 changed to a cysteine (MBP D95C) was also engineered (11). These MBP derivatives were expressed in E. coli TOP10 cells (Stratagene La Jolla, CA) by inducing with isopropyl β-D-1-thiogalactopyranoside (IPTG). Induction and expression was monitored by SDS

PAGE gel electrophoresis using 8/25% Phast gels stained with Coomassie Brilliant R Blue (Amerhsam Pharmacia, Piscataway NJ). Expressed protein was isolated using Ni-NTA agarose (Qiagen Valencia, CA) and dialyzed against phosphate-buffered saline (PBS) at pH 7.4 to remove excess imidazole. Protein concentration was determined from the absorbance at 280 nm using an Agilent 8453 UV-vis spectrophotometer (Walbronn, Germany). Purified dialyzed protein solution was sterile filtered with a 0.2 µM syringe filter and stored at 4 °C. MBP purified and stored in this manner remained functionally unchanged for at least 1 year (22). Protein Labeling. Maleimide and NHS (N-hydroxysuccinimide) ester mono- or bifunctionalized Cy3, Cy3.5, and Cy5 dyes were obtained from Amersham Pharmacia and the NHS ester of QSY9 from Molecular Probes (Eugene, OR). See Figure 2 for reactive dye structures and Table 1 for their photophysical properties. Approximately 1 mg of protein was reacted with NHSbifunctionalized Cy3 or Cy3.5 dye for 2.5 h at ∼pH 9.4 in the presence of 10 mM maltose. The MBP D95C mutant was labeled with monofunctional Cy3 maleimide after disulfide reduction by Cleland’s Reductacryl Reagent (Calbiochem). Labeled proteins were purified by gel filtration using PD-6 or PD-10 column chromatography (Amersham Pharmacia) to remove maltose and excess dye and then quantitated by spectrophotometry. The concentration of dye-labeled MBP was estimated using the formula:

[MBP-dye] ) (abs280 - (CF × absmax(dye)))/ (2) where CF is the correction factor for the absorbance of the dye at 280 nm (CF for Cy3 ) 0.08; CF for Cy3.5 ) 0.24) and absmax(dye) is the absorption measured at the dye absorbance maximum. The molar extinction coefficient () value used for MBP was 69 000 M-1 cm-1. The concentration of bound dye present was estimated using:

[dye] ) absmax(dye)/(dye)

(3)

The molar extinction coefficients of the dyes are presented in Table 1. Estimation of final dye/protein (D/ P) ratios was made using the formula:

(D/P)final ) [dye]/[MBP-dye]

(4)

β-Cyclodextrin Adduct Synthesis and Purification. An 8-10-fold molar excess of 6-monodeoxy-6monoamino-β-cyclodextrin, β-CD (Sigma, St. Louis, MO), was reacted with either monofunctional NHS-Cy5 or NHS-QSY9 for 2.5 h in 0.136 M sodium borate buffer (pH 8.5). The β-CD-dye adduct and excess monoamino β-CD were then precipitated by adding three volumes of

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Figure 2. Chemical structure of β-CD-dye adducts and reactive dye intermediates. (A) β-CD-QSY9, (B) β-CD-Cy5, (C) bis-functional NHS-Cy3, (D) bis-functional NHS-Cy3.5, and (E) Cy3-maleimide. Table 1. Photophysical Properties of the Dyes Used

dye

λmax abs (nm)

λmax abs bounda (nm)

λmax em (nm)

λmax em bounda (nm)

extinction coefficient (M-1 cm-1)

quantum yield

Cy3 Cy3.5 Cy5 QSY9

550 581 649 562

556 575 645 565

570 596 670 NA

567 596 669 NA

150,000 150,000 250,000 86,000

>0.15b >0.15b >0.28b NAc

a Cy3-MBP, Cy3.5-MBP, B-CD-Cy5, B-CD-QS9. b For labeled proteins where the dye-to-protein ratio ) 2. c NA ) not applicable.

ethanol followed by holding the preparation at -20 °C for 1 h. The precipitate collected by centrifugation was washed four times with absolute ethanol, collected again by centrifugation (4 °C), and dried in a vacuum centri-

fuge. Dried pellets were solubilized in HE buffer (10 mM HEPES, 1 mM EDTA buffer, pH 7.0) and product separated from unreacted β-CD and underivatized dye on 12% acrylamide 1 x TBE gels. The product band was excised and the β-CD-dye adduct eluted by passive diffusion into HE buffer. The β-CD-dye adduct was then concentrated and desalted using an oligonucleotide purification cartridge (OPC, Applied Biosystems, Foster City, CA). Briefly, the dilute β-CD-dye adduct in HEPES buffer was passed over the OPC several times to promote full binding to the resin. The bound β-CD-dye adduct was washed with ∼35 mL of 25 mM triethylamine ammonium acetate (TEAA) buffer (pH 7) and then eluted from the OPC with 1 mL 50% acetonitrile. The eluted product solution was aliquoted into 1.5 mL microfuge tubes, dried under vacuum, and stored at -20 °C. The

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pure adducts were dissolved in PBS for quantitation and usage. Fluorimetry. Fluorescence measurements were made using a SPEX Fluorolog-2 Fluorimeter (Jobin Yvon/ SPEX, Edison, NJ) with excitation at 520 nm and emission monitored at right angles with slit widths set at 2-5 nm. All measurements were made at 22 °C. Titrations of dye-labeled MBP and β-CD-dye adducts were carried out in 3 mL of PBS. Samples were allowed to equilibrate under constant stirring at least 5 min between each reagent/sample addition. Reagent/sample addition altered the final volume by less than 5%. Where indicated, fluorescence data was corrected for inner filter effects using the formula (5):

Fcorrd ) Fobsd × antilog(ODex + ODem/2)

(5)

where F is fluorescence and ODex and ODem are the optical density at the wavelength of excitation and emission, respectively. Apparent dissociation values (Kapp) were approximated from the corrected untransformed data using the methodology of Bagshaw and Harris (23). Values correspond to the approximate dissocation constants of maltose from dye-labeled MBP under the conditions of multiple equilibria that exist in these experiments. Mass Spectral Analysis. Mass spectral characterization of the β-cyclodextrin-dye adducts was performed using an Applied Biosystems API QSTAR Pulsar Mass Spectrometer by positive electrospray ionization (ESI). Samples were diluted in a solution of water:acetonitrile: glacial acetic acid (50:50:1) and infused into the spectrometer at a rate of 5 µL/min. The final concentrations of the β-cyclodextrin-QSY9 and β-cyclodextrin-Cy3 adducts used for ESI infusion were 2.5 and 10 µM, respectively. Intact MBP and MBP-Cy3.5 were initially analyzed using sinapinic acid as the ionization matrix. For mapping the location of dye-derivitized lysine residues in MBP, tryptic digests of MBP-Cy3.5 (D/P of 1.7) were subjected to MALDI-TOF/MS peptide analyses by Commonwealth Biotechnologies, Inc. (Richmond, VA). Peptide maps from MBP-Cy3.5 were compared to those from unmodified MBP, and a unique singly labeled peptide was identified based on theoretical vs actual mass for derivitized peptides. For the peptide mapping, ∼1.25 nmol of each protein was digested with trypsin. Digested samples were analyzed using cyano-4-hydroxycinnamic acid as the ionization matrix. Spectra were acquired in both reflector and linear modes. RESULTS

Mass Spectral Characterization of the β-Cyclodextrin-Dye Adducts and Mapping of Dye-Labeled MBP Lysine Residues. The results of mass spectral characterization of pure β-cyclodextrin-dye adducts: β-cyclodextrin-QSY9 (C81H104 N4O44S3) predicted molecular mass:1932.2, observed:1932.5; β-cyclodextrin-Cy5 (C75H109N3O14S2) predicted molecular mass:1771.6, observed:1771.4. The mass spectrum of the unmodified undigested MBP corresponded to both a single charged (m/z 41684) and doubly charged species (m/z 20828). The mass spectrum of the undigested MBP-Cy3.5 corresponded to the presence of the Cy3.5-labeled form (m/z 42694). Comparison of the mass spectra of tryptic digests for the unmodified and Cy3.5-modified MBPs identified a fragment (m/z 4660.92) in the modified MBP with three missed lysine cleavage sites. This mass corresponds to a singly-Cy3.5-

labeled peptide fragment (residues 139-171) with three missed lysine cleavage sites (residues 141, 143, 145). This strongly suggests that, for any derivatized MBP molecule, one of these lysines was labeled, although from the MS data we cannot unambiguously determine which one. Each of these lysines is located at the protein surface within one turn at the end of the same helix, and since the D/P ratio for the labeled protein is relatively low, it appears that for any single protein molecule one of these lysines undergoes strong preferential labeling, as opposed to the labeling occurring randomly, by reaction with the NHS ester of the dye. As an alternative, and to contrast a sensor assembly with completely unambiguous labeling at a unique site in MBP, we also utilized a maleimide labeling strategy to target the MBP 95 cysteine residue for Cy3 conjugation (D/P for D95C-Cy3 labeled protein was 1). This labeled protein was also investigated in a sensing role, as discussed below. The absorption and emission spectra of MBP Cy3 and MBP-Cy3.5 (both proteins labeled with amine reactive NHS esters of the dyes) are shown in Figure 3. Conjugating these dyes to MBP only slightly altered their absorbance (λmax abs) and emission (λmax em) maxima (Table 1). The largest spectral shift for protein bound vs free dye observed was for MBP-Cy3 and MBP-Cy3.5, both of which had their absorption maxima shifted ∼6 nm, with the MBP-Cy3 shifting to higher wavelength, and that of the MBP-Cy3.5 shifting to shorter wavelength. The identical shift in λmax emission was noted when MBP was labeled at 95C with Cy3-maleimide or on a lysine with amino-reactive Cy3. Assembly of the FRET Complex. As illustrated by the schematic in Figure 1, when a Cy3.5 dye-labeled MBP energy donor moiety binds a Cy5-labeled β-CD energy acceptor moiety, FRET should result, assuming the appropriate spectral overlaps and acceptable spacing between the dye centers (The same applies to the Cy3/ QSY9 pair). Figure 4 presents fluorescence emission spectra of a titration of 0.1 µM MBP-Cy3.5 FRET donor (1 D/P ratio) with increasing amounts of β-CD-Cy5 (FRET acceptor). Comparison of the fluorescence emission of each component measured separately and when both components were mixed shows the expected FRET effect. This effect is more apparent at the higher β-CDCy5 concentrations of 0.5 µM and 1 µM as demonstrated by the loss in donor intensity and the corresponding gain in acceptor fluorescence intensity. This intensity gain is much greater than the directly excited signal elicited from the identical concentration of uncomplexed β-CDCy5, shown for comparison in the same plots. Incubating 0.1 µM MBP-Cy3.5 with 1 µM of free Cy5 dye as a control resulted in an emission spectrum essentially identical to that of the independent MBP-Cy3.5 overlayed upon 1 µM independent β-CD-Cy5 shown in Figure 4, Panel C (Data not shown). Figure 5A shows the titration of 0.1 µM MBP-Cy3.5 (1.7 D/P) titrated with increasing amounts of β-CD-Cy5. This MBP-Cy3.5 preparation had a somewhat greater D/P ratio than the labeled protein used to derive the data shown in Figure 4. Stepwise addition of the acceptor portion caused a significant loss in donor fluorescence intensity almost identical to the pattern observed for MBP-Cy3.5 with the lower D/P ratio of 1 (Figure 4). This loss in donor fluorescent intensity is demonstrated by the plot in Figure 5B; an 81% drop in fluorescence intensity occurred at the wavelength corresponding to the donor’s emission maximum (596 nm) as a function of β-CD-Cy5 concentration. The observed shift to longer wavelength of β-CD-Cy5 emission, ∼6-8 nm, shown in Figure 5A,

FRET Maltose Sensor

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Figure 3. Absorption and emission spectra of conjugated MBP-Cy3.5 (1 dye/protein ratio), solid line, and β-CD-Cy5, dotted line (A). MBP-Cy3 (1.7 dye/protein ratio), solid line, and β-CD-QS9, dotted line (B). For emission spectra, samples were excited at 520 nm. Data are normalized for presentation. Arrows indicate the axis of reference.

is indicative of inner filter effects at higher reagent concentrations (25). Control experiments where 0.1 µM MBP-Cy3.5 (1 D/P) was titrated with unlabeled β-CD (Figure 5C) resulted in no change in donor fluorescence. The converse control experiment where 500 nM of β-CDCy5 was titrated with increasing amounts of unlabeled MBP also showed no change in acceptor emission (Figure 5D). Additionally, in a control experiment in which dyelabeled MBP was titrated with maltose there was no effect on fluorescence (data not shown). Taken together, these results demonstrate that a FRET process is indeed taking place between the donor and acceptor components of the homogeneous sensing complex. Maltose Analysis. Figure 5E presents plotted data from the titration of the assembled FRET complex (0.1 µM MBP-Cy3.5 (1.7 D/P) - 2.5 µM β-CD-Cy5) with increasing concentrations of maltose. Increasing the maltose concentration causes a systematic increase in MBP-Cy3.5 donor fluorescence intensity. The corrected fluorescence intensities as a function of added maltose are plotted in Figure 5F. An increase of 76% in Cy3.5 donor fluorescence intensity is observed for the completed titration. A similar increase in donor intensity was seen for the 0.1 µM MBP-Cy3.5 (1 D/P) - 2.5 µM β-CD-Cy5 FRET complex when it was titrated with increasing amounts of unlabeled β-CD (data not shown). From these data, Kapp values of 0.14 and 0.15 µM were estimated for titrations using MBP-Cy3.5 protein with 1.0 and 1.7 D/P ratios, respectively, see Table 2 (23). MBP-Cy3-β-CD-QSY9 FRET Ensemble. An alternate FRET complex consisting of Cy3 as the donor and

a dark quencher as energy acceptor was investigated. The dark quencher, QSY9, was conjugated to the β-CD and purified as described. MBP was labeled with Cy3, chosen as the energy donor in order to optimize donor-acceptor spectral overlap (see Figure 3, Panel B). This FRET pair functions in an analogous manner to the MBP-Cy3.5β-CD-Cy5 pair, but the dark quencher has no fluorescence emission. Figure 6A shows the titration of a 0.1 µM MBP-Cy3-5 µM β-CD-QSY9 FRET complex with increasing concentrations of maltose (MBP was labeled on lysine residues). The presence of the β-CD-QSY9 adduct at 5 µM alters the shape of the Cy3 emission spectra, in comparison to uncomplexed MBP-Cy3 (Figure 3B), due to inner filter effects (25). Nonetheless, the assembly functions well in sensing maltose. Data were corrected for inner filtering at 567 nm and plotted to demonstrate the net change in fluorescence intensity as a function of maltose concentration (Figure 6B), and a Kapp value of 0.82 µM was estimated for maltose for this sensor complex under these conditions (Figure 6C). MBP-D95C-Cy3-β-CD-QSY9 FRET Ensemble (1 D/P). To evaluate a sensor assembly where the site of donor labeling was more completely controlled, a FRET ensemble was prepared that included a variant of MBP engineered with a single cysteine for unique thioldirected labeling with a Cy3 maleimide derivative. In this case, when a 0.1 µM MBP-D95C-Cy3 to 1 µM β-CDQSY9 sensor assembly was titrated against increasing amounts of maltose (Figure 6C), a Kapp value of 2.90 µM was estimated for maltose binding. The maltose sensing behavior for this assembly was found to be comparable

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Figure 4. Titration of 0.1 µM MBP-Cy3.5 (1 dye/protein ratio) with increasing amounts of β-CD-Cy5; 0.1 µM in A, 0.5 µM in B, and 1 µM in C. In each panel the fluorescence of the 0.1 µM MBP-Cy3.5 uncomplexed (solid line), the β-CD-Cy5 uncomplexed (dotted line), and the assembled FRET complex (solid squares) are shown. Samples were excited at 520 nm.

to the above examples in which the protein was modified with the Cy3 energy donor by attachment to the  amino group of lysine residues that occurred in a fortuitously selective manner in the area of residues 141-145 of MBP. DISCUSSION

Wild-type, unlabeled MBP has a reported KD of 1.8 µM for β-CD and 0.9 µM for maltose (11, 24). Since these values are similar, the appropriate concentration of maltose could compete with dye-labeled β-CD for binding to the protein in the present sensor assemblies. In these sensors, displacement of the labeled β-CD translates to loss of FRET and to an increase in donor emission. This allows measurement of maltose concentrations through the gain in donor fluorescence intensity and demonstrates that the FRET complexes can function as quantitative homogeneous biosensors. The FRET complexes are quite sensitive, with lower detection limits of 50100 nM maltose and upper limits approaching 50 µM (Figure 5E and Figure 6A). These assemblies are more sensitive than the previously described FRET MBP sensor that detected maltose in only the micromolar

range (3). This is likely due to the Cy3 and Cy3.5 dyes being better energy transfer donors than endogenous tryptophan residues, as well as the large extinction coefficients and high quantum yields of cyanine dyes. Structural modeling of the present sensor assemblies indicates that the distance between the bound β-CDdyes and any of the labeled donor dyes covalently attached to the protein surface will be in the 29-45 Å range. From the MBP crystal structure, the distance for each of the mapped Cy3.5 derivitized lysines to the β-CD-dye located in the binding pocket is as follows: lysine 141, ∼40 Å; lysine 143, ∼46 Å; lysine 145, ∼45 Å. The Fo¨rster distance corresponding to 50% energy transfer efficiency (R0) for the Cy3.5-Cy5 FRET pair based on spectral overlap is 66.2 Å. In the 29-45 Å range, the energy transfer efficiency increases to 91 to 98%, respectively. From the close proximity of each of these lysines, and their very similar distances from the sugar binding pocket (well under R0), it is clear that labeling at any one of these residues will result in effective FRET-based sensing. The R0 for the Cy3-QSY9 FRET donor-acceptor pair is 54.8 Å, and the predicted FRET efficienies for this

FRET Maltose Sensor

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Figure 5. (A) Fluorescence emission spectra of 0.1 µM MBP-Cy3.5 (1.7 dye/protein ratio) titrated against increasing amounts of β-CD-Cy5. (B) Reduction in fluorescence intensity (uncorrected) at 596 nm for the titration shown in A. (C) Fluorescence emission spectra of 0.1 µM MBP-Cy3.5 (1 dye/protein ratio) titrated with increasing amounts of unlabeled β-CD. (D) 0.5 µM β-CD-Cy5 titrated with increasing amounts of unlabeled MBP. (E) FRET donor’s emission spectra during a titration of 0.1 µM MBP-Cy3.5 (1.7 dye/protein ratio) mixed with 2.5 µM β-CD-Cy5 against increasing amounts of maltose. (F) Gain in fluorescence intensity (corrected) for the titration shown in E. Samples were excited at 520 nm. Table 2. Properties of MBP-Dye Conjugates Used MBP-dye conjugate

MBP protein-dye ratio

apparent maltose dissociation values Kapp (µM)

MBP-D95C-Cy3 MBP-Cy3 MBP-Cy3.5 MBP-Cy3.5

1.0 1.7 1.0 1.7

2.90 0.82 0.14 0.15

pair at the 29-45 Å distance range are 98% and 77%, respectively (24, 26). The ∼25 Å distance between Cy3 located at 95C and β-CD-dye bound in the MBP sugar

binding pocket corresponds to a predicted FRET efficiency approaching 100%. Clearly, the donor-acceptor distances in each of these complexes are ideal for sensing by the FRET mechanism to occur. X-ray crystal structure studies and structural modeling demonstrate that while MBP binds β-CD and remains in an open conformation, it closes by an intramolecular conformational change so as to engulf maltose (11, 24). Previously described MBP-based sensors have exploited the conformational changes that MBP undergoes when binding maltose by measuring the effects of this allosteric

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Figure 6. (A) Fluorescence emission spectra of 0.1 µM MBP-Cy3 (1.65 D/P) complexed with 5 µM β-CD-QSY9 and titrated against increasing amounts of maltose. (B) Corrected fluorescence intensity at 567 nm plotted against maltose concentration. (C) Plot of the corrected fluorescence intensity at 1/2 fluorescence max that was used to estimate the apparent dissociation value, Kapp (23). (C) Titration of 0.1 µM MBP95C-Cy3 complexed to 1 µM β-CD-QSY9 against increasing amounts of maltose.

change upon intrinsic tryptophan fluorescence or on the fluorescence emanating from dye-labeled MBP (11). Indeed, some data suggest that fluorophore conjugation to certain MBP mutants may stabilize the maltose-bound form of the protein (11). In contrast, titrating MBPCy3.5 with maltose or unlabeled β-CD results in no fluorescence emission changes even at saturating ligand concentrations. Thus, the results presented here demonstrate that allosteric effects are silent in the present sensor complexes and that the observed sensing behavior is solely due to FRET. These sensing assemblies make use of novel fluorescent dye-labeled β-CD adducts. Other activated dye labels

could undoubtedly be attached to monoamino β-CD to expand the potential utility of the assay. As we have shown, the basic FRET sensor system described can function in both fluorescent donor-fluorescent acceptor and fluorescent donor-dark quencher configurations. Additionally, the Cy3.5-Cy5 donor-acceptor configuration functions almost identically at two different dye/ MBP ratios (1 and 1.7). This suggests that controlling the exact D/P ratio is not critical for sensor performance. The dye-labeled MBP-β-CD-QSY9 FRET pair is particularly appealing since a fluorescence increase or ‘switching on’ occurs in the presence of maltose, with the added benefit of no overlapping acceptor emission.

FRET Maltose Sensor

Apparent dissociation (KD,app) values were estimated from these sensor complexes for maltose and were very close to previous reported maltose Kd values. For example, Kapp ) 0.82 µM for a MBP-Cy3 protein versus 0.8 µM for the acrylodan-modified C95 mutant protein (3). A Kapp value of 2.90 µM was determined for the MBP-D95C Cy3-labeled mutant/QSY9-β-CD system, which correlates well with a value of 1.5 µM for the same mutant in an unlabeled form and 4.4 µM when the mutant is labeled with IANBD at that site (11). The 0.14 and 0.15 µM values estimated for the MBP-Cy3.5/β-CDdye systems are essentially identical to the KD of 0.1 µM described for the MBP Ile329Cys mutant modified with an IANBD fluorescent reporter (11). The MBP sugar receptor used in the present MBPbased maltose sensors functioned effectively when either labeled with an energy donor in a fully directed manner (specific cysteine labeling) or when labeled with lysinedirected dye derivatives. Although labeling with the lysine-directed dye derivatives was fortuitously selective in the present case, even essentially randomly labeled protein would be highly likely to function effectively in these FRET configurations due to the moderate size of MBP, the central placement of its sugar binding site, and the favorable R0 values selected for donor-acceptor pairs. Potential applications of these sensor complexes include direct use of their maltose sensing capabilities in food production and processing (16), specifically in beer and bread production which utilize maltose as the primary sugar source and potentially other fermentative technology. Additionally, variants of this sensing scheme may be adapted for glucose monitoring of diabetic patients (8, 25) or for sensing of surface glycoproteins of potential antigens (27). ACKNOWLEDGMENT

The authors thank Prof. Homme Hellinga (Duke University) for providing the plasmid with the MBPHIS-tagged gene sequence utilized. I.L.M. was supported by a National Research Council (NRC) fellowship through the Naval Research Laboratory. M.E.L. was supported by an American Society for Engineering Education (ASEE) fellowship. The views, opinions, and/or findings described in this report are those of the authors and should not be construed as official Department of the Navy positions, policies, or decisions. LITERATURE CITED (1) Iqbal, S. S., Mayo, M. W., Bruno, J. G., Bronk, B. V., Batt, C. A., and Chambers, J. P. (2000) A Review of Molecular Recognition Technologies for Detection of Biological Threat Agents. Biosens. Bioelect. 15, 549-578. (2) Ligler F. S., and Rowe Taitt, CA C. A. Eds. (2002) Optical Biosensors: Present and Future. Elsevier, The Netherlands. (3) Gilardi G., Zhou L. Q., Hibbert L., and Cass A. E. G. (1994) Engineering the Maltose Binding Protein for Reagentless Fluorescence Sensing. Anal. Chem. 66, 3840-3847. (4) Didenko V. V. (2001) DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications. BioTechniques 31, 1106-1121. (5) Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Academic/Plenum Press, New York. (6) Tong, A. K., Zengmin, L., and Jingyue, J. (2002) Combinatorial Fluorescence Energy Transfer Tags: New Molecular Tools for Genomics Applications. IEEE J. Quantum Electron. 38, 110-121. (7) Schobel, U., Egelhaaf, H.-J. Brecht, A., Oelkrug, D., and G. Gauglitz. (1999) New Donor-Acceptor Pair for Fluorescent

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