Single-Molecule Assays of Calmodulin Target Binding Detected with a

E. Shane Price , Matthew S. DeVore and Carey K. Johnson ... Ian Miller , Ruslan Sanishvili , Christopher M. Jenkins , Richard W. Gross , Sergey Korole...
0 downloads 0 Views 394KB Size
Download PDF
Anal. Chem. 2004, 76, 3630-3637

Single-Molecule Assays of Calmodulin Target Binding Detected with a Calmodulin Energy-Transfer Construct Michael W. Allen,† Ramona J. Bieber Urbauer,‡,§ and Carey K. Johnson*,†

Department of Chemistry and Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

We have detected single-molecule binding interactions of a target peptide with the calcium-signaling protein calmodulin (CaM) immobilized in an agarose gel, and we have demonstrated the application of a single-molecule binding assay to measure the binding strength of CaM with the CaM-binding domain of calmodulin-dependent protein kinase II (CaMKII). The results demonstrate the potential for ultrasensitive assays of CaM-target interactions and the measurement of a picomolar dissociation constant. To detect single-molecule protein interactions, singlemolecule assays require that the analyte molecule be confined to the focal spot of the objective for the time scale of the measurement. We demonstrate the deleterious effect of surface immobilization on CaM. As an alternative to surface immobilization, we have constructed a CaM/ maltose binding protein fusion protein, which renders CaM translationally immobile in a low weight percent agarose gel. The target binding functionality of CaM assayed in agarose gels is in good agreement with solution assays. The utility of the construct for detecting interactions with CaM targets was demonstrated in a singlemolecule assay of binding interactions of MBP-CaM with the CaMKII CaM-binding domain peptide. A value of 103 ( 35 pM for the dissociation constant of this interaction was determined by simple counting of fluorescent molecules.

hundred molecules. Single-molecule spectroscopy further provides a powerful methodology for revealing subpopulations frequently masked by the ensemble averaging of conventional fluorescence spectroscopic methods.6-10 Assays performed via single-molecule fluorescence techniques can reveal interaction parameters such as binding constants on concentration regimes that are not obtainable by conventional ensemble methods. The potential coupling of single-molecule fluorescence detection with highthroughput methodologies would permit rapid, highly sensitive assays of protein interactions with minimal sample quantities.11-13 To develop assays at the single-molecule level, two important issues must be addressed. First, monitoring the fluorescence of a single molecule for an extended period of time requires that the molecule be confined to the focal volume of a high-numericalaperture objective lens for the time scale of the measurement. Second, the biological function, e.g., target binding, of the immobilized molecule must not be significantly altered by the immobilization scheme. Many binding assays performed on both the single-molecule and ensemble levels immobilize analyte molecules on surfaces. Careful attention must be given to the possibility that surface immobilization may drastically alter the behavior of immobilized molecules. There is therefore a need to identify immobilization methods that do not impair protein function. In contrast to surface immobilization, an agarose gel matrix allows single-molecule assays of proteins immobilized away from a solid support.7,14 Because a matrix of small pores15 is formed

As the fields of analytical chemistry and molecular biology converge, the development of new methods to assay protein interactions is paramount. In biological systems, any given protein may interact with numerous other proteins, peptides, or other small molecules (hormones, drug molecules, etc.), intensifying the challenge of identifying and characterizing the strength of these interactions. The most sensitive biochemical assays employ fluorescence detection. However, conventional fluorescence assays still require analyte concentrations on the order of tens of nanomolars. In recent years, ultrasensitive detection has been achieved through single-molecule fluorescence spectroscopy,1-5 which can be used to detect and extract information from a few

(1) Peck, K.; Stryer, L.; Glazer, A. N.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 4087-4091. (2) Soper, S. A.; Shera, E. B.; Martin, J. C.; Jett, J. H.; Hahn, J. H.; Nutter, H. L.; Keller, R. A. Anal. Chem. 1991, 63, 432-437. (3) Nie, S.; Chiu, D. T.; Zare, R. N. Anal. Chem. 1995, 67, 2849-2857. (4) Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Orden, A. V.; Werner, J. H.; Keller, R. A. Chem. Rev. 1999, 99, 2929-2956. (5) Ma, Y.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2000, 72, 4640-4645. (6) Moerner, W. E.; Orrit, M. Science 1999, 283, 1670-1676. (7) Lu, H. P.; Xun, L.; Xie, X. S. Science 1998, 282, 1877-1882. (8) Keller, R. A.; Ambrose, W. P.; Arias, A. A.; Cai, H.; Emory, S. R.; Goodwin, P. M.; Jett, J. H. Anal. Chem. 2002, 74, 316A-324A. (9) Weiss, S. Science 1999, 283, 1676-1683. (10) Xie, X. S.; Trautman, J. K. Annu. Rev. Phys. Chem. 1998, 49, 441-480. (11) Anazawa, T.; Matsunaga, H.; Yeung, E. S. Anal. Chem. 2002, 74, 50335038. (12) Auroux, P.-A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (13) McDonald, J. C.; Whitesides, G. M. Acc. Chem. Res. 2002, 35, 491-499. (14) Brasselet, S.; Peterman, E. J. G.; Miyawaki, A.; Moerner, W. E. J. Phys. Chem. B 2000, 104, 3676-3682. (15) Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A. Biophys. J. 1999, 77, 542552.

* Corresponding author. Phone: 785-864-4219, Fax: 785-864-5396, E-mail: [email protected]. † Department of Chemistry. ‡ Department of Molecular Biosciences. § Present address: Department of Chemistry and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602.

3630 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

10.1021/ac0497656 CCC: $27.50

© 2004 American Chemical Society Published on Web 05/07/2004

at a very low weight percent of agarose, the environment surrounding the immobilized molecule is up to 99% biological buffer. In this paper, we examine the function of calmodulin (CaM) immobilized at a surface and in agarose gels. CaM is known to bind over 30 target molecules including phosphatases, kinases, receptors, channels, and pumps.16-18 These interactions involve CaM in numerous intra- and extracellular signaling pathways. There is thus a need for sensitive assays of the interactions of CaM with its diverse targets and ligands.19,20 One such target of CaM is the calmodulin-dependent protein kinase II (CaMKII). CaMKII is present in many tissues, primarily the brain, and plays a role in many cellular functions. CaMKII is a mediator of calcium neuronal signaling and is essential in processes such as neurotransmitter synthesis, gene expression, and long-term potentiation associated with long-term memory.21,22 The 281-309-residue peptide of CaMKII has been shown to bind CaM with a high affinity,23,24 making the characterization of binding difficult by conventional ensemble methods. We have designed a single-pair energy-transfer construct that is capable of probing the binding of peptide and drug targets to CaM. Resonance energy transfer from the donor dye to the acceptor dye allows detection of fluorescence from the acceptor dye at a wavelength tens of nanometers away from the excitation wavelength. This increases the signal-to-background ratio by decreasing the contribution to the background from scattered excitation light and Raman scattering by water in the agarose gel matrix. Additionally, strong quenching of the donor or acceptor dye occurs upon target binding only when both dyes are present. We demonstrate the loss of target binding functionality when CaM is immobilized on a glass surface through both many-molecule and single-molecule assays. These results demonstrate the necessity to evaluate surface interactions for molecules immobilized at or near a surface. We present an assay that demonstrates the retention of target-binding functionality of CaM when immobilized in an agarose gel matrix, in contrast to CaM immobilized on a glass surface. We have previously found that CaM itself is not immobilized in agarose gels.25 Therefore, a fusion construct was designed where the N-terminal domain of CaM is fused to maltosebinding protein (MBP) by a 13-amino acid linker.25 The fusion of CaM to MBP renders the construct translationally immobile in agarose gels.25 Using a known CaM antagonist, amitriptyline, we observe nearly identical binding constants for the CaM fusion protein in bulk solution and in agarose gels, further demonstrating the retention of target binding and the absence of interference (16) Chattopadhyaya, R.; Meador, W. E.; Means, A. R.; Quiocho, F. A. J. Mol. Biol. 1992, 228, 1177-1192. (17) Crivici, A.; Ikura, M. Annu. Rev. Biophys. Biomol. Struct. 1995, 24, 85116. (18) Trewhella, J. Cell Calcium 1992, 13, 377-390. (19) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 2101-2105. (20) Douglass, P. M.; Salins, L. L. E.; Dikici, E.; Daunert, S. Bioconjugate Chem. 2002, 13, 1186-1192. (21) Colbran, R. J.; Soderling, T. R. Curr. Top. Cell. Regul. 1990, 31, 181-221. (22) Bayer, K. U.; Schulman, H. Biochem. Biophys. Res. Commun. 2001, 289, 917-923. (23) Payne, M. E.; Fong, Y. L.; Ono, T.; Colbran, R. J.; Kemp, B. E.; Soderling, T. R.; Means, A. R. J. Biol. Chem. 1988, 263, 7190-7195. (24) To ¨ro ¨k, K.; Tzortzopoulos, A.; Grabarek, Z.; Best, S. L.; Thorogate, R. Biochemistry 2001, 40, 14878-14890. (25) Allen, M. W.; Urbauer, R. J. B.; Zaidi, A.; Williams, T. D.; Urbauer, J. L.; Johnson, C. K. Anal. Biochem. 2004, 325, 273-284.

from the agarose gel matrix. The ability to immobilize CaM by a fusion construct with MBP suggests that a variety of small proteins or peptides could also be immobilized in a gel by fusion with MBP. MBP possesses several properties that make it a suitable fusion partner: it has no cysteine residues that would be modified by fluorescent probes,26 it does not oligomerize at micromolar concentrations,26 it is stable at the slightly elevated temperatures required to form the gel,27 and it has commercially available expression vectors and established purification protocols to simplify the expression of MBP in Escherichia coli.26,28 We have used the CaMKII peptide as a model system to study CaM target binding. Using the quenching of acceptor fluorescence upon the binding of CaMKII peptide by CaM, we have assayed the binding activity of CaM in a many-molecules experiment and on the singlemolecule level. By performing assays with single-molecule sensitivity, we have directly measured the picomolar binding constant of the CaMKII peptide fragment with CaM. MATERIALS AND METHODS Materials. pET-15b and E. coli competent cell strain BL21(DE3) were purchased from Novagen (Madison, WI). All other restriction and modifying enzymes, reagents, and competent cells used for standard molecular biology procedures were purchased from Invitrogen (Carlsbad, CA). Phenyl Sepharose CL-4B resin was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). DNA sequencing was performed at the University of Kansas Biochemical Research Service Laboratory. The fluorescent probe conjugation was performed with maleimide derivatives of AlexaFluor 488 and Texas Red (Molecular Probes). HPLC grade acetonitrile was purchased from Fisher Scientific, and trifluoroacetic acid (99%) was purchased from Acros Organics. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Spectroscopy. A Varian Bio 100 UV/visible spectrometer was used to collect all absorption spectra, and a Varian Eclipse fluorometer was used to collect all emission spectra. The resolution of the absorption and emission spectra was (5 nm. ESI mass spectra were acquired on a Q-TOF-2 (Micromass Ltd., Manchester, U.K.) hybrid mass spectrometer operated in MS mode and acquiring data with a time-of-flight analyzer. Expression and Purification of Protein Constructs. Threonine residues 34 and 110 of wild-type chicken CaM were altered to cysteine by site-directed mutagenesis (T34C,T110C-CaM). A fusion protein of the double-cysteine mutant was constructed with MBP tethered to the N terminus of CaM via a 13-residue linker (denoted MBP-T34C,T110C-CaM), as previously described.25 The details of the preparation of T34C,T110C-CaM are given in the Supporting Information and are similar to those for the preparation of the fusion construct. Labeling and Purification of Fluorescently Labeled Constructs. Purified T34C,T110C-CaM and MBP-T34C,T110C-CaM were labeled simultaneously with both donor (AlexaFluor 488) and acceptor (Texas Red) fluorescent probes in a manner previously described.25 The protocol for the fluorescent labeling of T34C,T110C-CaM is given in Supporting Information. (26) Kellermann, O. K.; Ferenci, T. Methods Enzymol. 1982, 90, 459-463. (27) Ganesh, C.; Shah, A. N.; Swaminathan, C. P.; Surolia, A.; Varadarajan, R. Biochemistry 1997, 36, 5020-5028. (28) Bedouelle, H.; Blondel, A.; Bregegere, F.; England, P.; Nageotte, R.; Rondard, P. Perspectives on Protein Engineering & Complementary Technologies, Collected Papers, International Symposium, 3rd, Oxford, September 13-17, 1994, 1995; pp 180-183.

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

3631

All chromatography was performed on a Waters HPLC 600 system equipped with a model 2487 dual-wavelength absorption detector. A C18 column (Vydac 218TP54) consisting of 5-µm particles with an average pore size of 300 Å was used for the isolation of the donor- and acceptor-labeled fusion CaM (MBP‚ CaM-DA). The best purification of donor- and acceptor-labeled CaM (CaM-DA) was obtained using a C5 column (Supelco Discovery BIO Wide Pore 567231-U) consisting of 3-µm particles with an average pore size of 300 Å. AlexaFluor 488 absorption was detected at its maximum absorption wavelength of 493 nm, and Texas Red was detected at its maximum absorption wavelength of 593 nm. The UV absorption of the aromatic side chains at 280 nm was used to monitor the unlabeled protein. Immobilization of CaM-DA. Single CaM-DA molecules were immobilized on a glass cover slide by spin coating. Glass cover slides were cleaned by immersing them in methylene chloride and rinsing them with deionized-distilled (18 MΩ) water. The cover slides were then immersed in an acid cleaning bath containing 1 mM ammonium peroxydisulfate in concentrated sulfuric acid, rinsed with deionized-distilled water, immersed in a bath containing potassium hydroxide dissolved in isopropyl alcohol, and rinsed with deionized-distilled water. A minimum of three cycles of the acid and base cleaning solutions and rinsings were performed. Clean slides were stored under ambient conditions, rinsed again with deionized-distilled water, and dried with nitrogen prior to use. For the spin-coating procedure, a small volume (10-20 µL) of the CaM-DA solution was allowed to bind to the glass surface for 5-10 s and then rotated at 5000 rpm for several seconds to remove any excess solution. All single-molecule experiments using surface-immobilized molecules were performed with a physiological buffer solution, consisting of 10 mM HEPES, pH 7.2, 0.1 M NaCl, 1.0 mM MgCl2, and 0.1 mM CaCl2, placed on top of the glass surface. A stainless steel washer, flattened on one side by machining, was used to confine buffer solutions above the cover slide. Up to 80 µL of solution was placed inside the washer, and a second cover slide was placed on top of the washer to slow evaporation. In this configuration, experiments could be performed for extended periods of time before the buffer evaporated from the surface. MBP‚CaM-DA was immobilized in low weight percent agarose gels prepared with type VIII-A low gelling temperature agarose (Sigma). For amitriptyline titration ensemble measurements, bulk gels were prepared by diluting a concentrated solution of MBP‚ CaM-DA with a 2.0% (w/w) agarose gel solution and the physiological buffer solution. The final concentration of MBP‚CaM-DA was 100 nM, and the final agarose gel concentration was 1.5% (w/w). To prepare gels for single-molecule experiments, 15 µL of a 3.0 nM solution of MBP‚CaM-DA was mixed with 80 µL of a 1.75% (w/w) solution of warm agarose to yield a final agarose concentration of 1.5% (w/w) and a final MBP‚CaM-DA concentration of 475 pM. Gels for the CaMKII binding experiments were prepared by placing a small volume of warm gel solution (15 µL) between two cover slides. These gels could be examined for as long as 2 h before the gel matrix deteriorated due to water evaporation. Single-Molecule Fluorescence Microscopy and Image Analysis. The dual-channel single-molecule fluorescence microscope has been described in detail elsewhere.25 For these experiments, the 488-nm line of an Ar+/Kr+ laser was used for excitation of the donor fluorophore, AlexaFluor 488. The beam 3632

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

was expanded and collimated by a telescope and reflected off a dichoric mirror completely backfilling a 1.3-NA 100× objective (Nikon CFI Superfluor). The fluorescence emission from the sample was collected by the same objective, passed through the dichoric mirror, and directed out the side port of the microscope. A second dichroic mirror was used to split the fluorescence into donor and acceptor channels. Band-pass filters placed in front of the donor and acceptor single photon counting modules provided further discrimination of photons. The sample was rastered across the objective by a piezoelectric scanning stage (Mad City Labs, Nano H100). For the singlemolecule surface experiments, 10 µm × 10 µm (256 pixel × 256 pixel) images of the acceptor channel were acquired. For the determination of the CaMKII peptide binding constant, 20 × 20 µm (256 pixel × 256 pixel) images were acquired with the excitation beam focused into the gel 10-12 µm above the glass surface. Focusing above the surface dramatically reduces the fluorescence contribution from MBP‚CaM-DA molecules located at the surface. A new gel was prepared for each CaMKII peptide concentration. For each 20 × 20 µm scan, two images were acquired, one for the trace and one for the retrace of each line. A minimum of 40 images were collected for each CaMKII peptide concentration and used for subsequent analysis. The images were converted from text files to bitmaps using the Scion Image software program (Scion Corp.).29 The bitmap images were used to determine the number of MBP‚CaM-DA molecules in each scan using Lispix software.30 For each image, the threshold was adjusted to exclude counts that resulted from background fluorescence or scatter. The “blob” counting tool of the Lispix program was then used to determine the number of single-molecule spots in each image. “Blobs” were identified by requiring a minimum of 10 adjacent pixels above the minimum threshold value, and blobs that overlapped the edge of the image were included. The validity of the blob counting method was verified by comparing to the number of molecules determined by visual counting in several sample images. The number of molecules determined by Lispix was within 5% of the number determined by visual inspection. RESULTS AND DISCUSSION Preparation and Characterization of CaM-DA and MBP‚CaM-DA. The level of expression and solubility of T34C,T110C-CaM is nearly identical to that previously reported for MBP-T34C,T110C-CaM and wild-type CaM.25 The fluorescent labeling reaction of T34C,T110C-CaM generates a mixture containing six possible species: unlabeled T34C,T110C-CaM, T34C,T110C-CaM labeled with one (CaM-D) or two (CaM-DD) donor dyes, T34C,T110C-CaM labeled with one (CaM-A) or two (CaM-AA) acceptor dyes, and T34C,T110C-CaM labeled with both donor and acceptor dyes (CaM-DA). The isolation of MBP‚CaMDA has been described previously.25 CaM-DA was separated from the other labeled species by a similar procedure. The identity of the purified construct was verified by ESI mass spectrometry. See Supporting Information for specific details and a complete dissection of the CaM-DA chromatogram. Bulk Fluorescence Emission Spectra of CaM-DA. A 0.1 µM solution of CaM-DA in the physiological buffer (see Materials (29) Program available from http://www.scioncorp.com/frames/fr_scion_products.htm. (30) Program available from http://www.nist.gov/lispix/.

Figure 1. (A) Fluorescence emission spectrum of a 0.1 µM CaM-DA solution excited at 488 nm, curve 1. Curves 2 and 3 show the emission spectra of a 0.1 µM solution of a single CaM mutant, CaM T34C, labeled with AlexaFluor 488 and Texas Red, respectively, excited at 488 nm. (B) Fluorescence emission spectrum of a 0.1 µM CaM-DA solution excited at 488 nm in the presence of 0.6 µM CaMKII peptide, curve 1. Fluorescence emission spectrum of a 0.1 µM CaM-DA solution excited at 488 nm, curve 2. (C) Curves 1 and 2 are the emission spectra of a 0.1 µM solution of T34C-CaM labeled with AlexaFluor 488 and Texas Red, respectively. Curves 3 and 4 are the emission spectra of a 0.1 µM solution of T34C-CaM labeled with AlexaFluor 488 and Texas Red, respectively, in the presence of 0.6 µM CaMKII peptide. Curves 1 and 3 were acquired with excitation at 488 nm; curves 2 and 4 were acquired with excitation at 580 nm.

and Methods) was excited at 488 nm and the fluorescence emission collected from 500 to 750 nm (Figure 1A, curve 1). The spectrum shows fluorescence from both AlexaFluor 488 and Texas Red, demonstrating energy transfer from AlexaFluor 488 to Texas Red. The emission spectrum of the single mutant T34C-CaM labeled with AlexaFluor 488 (0.1 µM) is shown in Figure 1A, curve 2 for comparison. To demonstrate that the emission of Texas Red is minimal as a result of direct excitation at 488 nm, the emission spectrum of T34C-CaM labeled with Texas Red (0.1 µM) is shown in Figure 1A, curve 3. In binding a peptide, the structure of CaM changes from an extended dumbbell shape into a compact structure that wraps around the peptide.16 This structure brings the donor and acceptor dyes of CaM-DA into proximity, resulting in fluorescence quenching. The fluorescence emission spectrum of 0.1 µM CaM-DA incubated with a saturating amount (0.6 µM) of CaMKII peptide in the physiological buffer (Figure 1B, curve 1) shows that, upon binding of the CaMKII peptide, the fluorescence of both the donor and acceptor dyes is quenched by nearly 75%. (For comparison, the emission spectrum of CaM-DA in the absence of CaMKII peptide is shown again in Figure 1B, curve 2.) For an interaction between donor and acceptor fluorophores described by Fo¨rster resonance energy transfer, one would expect the proximity of the two dyes in the CaM-DA-peptide complex to result in quenching of donor emission with a concomitant enhancement of acceptor emission. What we observe, however, is quenching of emission of both fluorophores (Figure 1B, curve 1). The strong quenching of both fluorophores suggests a close interaction between donor and acceptor dyes that results in quenching of acceptor emission. In contrast, the fluorescence emission of a single cysteine CaM mutant, T34C-CaM labeled with a single donor fluorophore (AlexaFluor 488) (Figure 1C, curve 1) or a single acceptor

fluorophore (Texas Red) (Figure 1C, curve 2), is quenched by only 2 and 11% (Figure 1B, curves 3 and 4), respectively, in the presence of a saturating amount (0.6 µM) of CaMKII peptide. Thus, target binding results in little quenching of AlexaFluor 488 or Texas Red in the absence of the other dye. Identical quenching was observed when the fluorophores were placed on the other cysteine, T110C-CaM (data not shown). The small fluorescence intensity change in the singly labeled constructs upon peptide binding is not sufficient to generate a sensitive, quantifiable change in fluorescence, whereas the quenching of both fluorophores upon target binding to CaM-DA results in a readily detectable drop in fluorescence intensity. The dramatic quenching of CaM-DA acceptor emission signals peptide target binding by CaM in a manner detectible by single-molecule spectroscopy. CaM-DA Immobilized on a Glass Surface Shows No Peptide Target Binding Activity. In many assays, biomolecules are immobilized on a substrate surface and analyte solutions are passed over the solid support for determination of target binding.11,31,32 While detection can take many forms, a change in fluorescence intensity is frequently employed due to its sensitivity. Using the change in fluorescence intensity of the acceptor dye upon binding the CaMKII peptide, immobilized CaM-DA on a glass surface would provide a convenient method of quantifying the steady-state binding equilibrium. We show here, however, that surface-immobilized CaM-DA does not retain target-binding functionality. For an initial assessment of the activity of CaM-DA immobilized on a glass surface, “subensemble” measurements were carried out with a solution of CaM-DA in a physiological buffer spin-coated (31) Gaspar, S.; Schuhmann, W.; Laurell, T.; Csoregi, E. Rev. Anal. Chem. 2002, 21, 245-266. (32) Taylor, R. F. Handb. Chem. Biol. Sens. 1996, 203-219.

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

3633

Figure 2. (A) Integrated intensity of CaM-DA molecules spin-coated on a glass cover slide excited at 488 nm. The bars show the intensity from spin-coated CaM-DA molecules with a physiological buffer placed on top of the surface; spin-coated CaM-DA molecules covered with a buffer containing 0.6 µM CaMKII peptide; CaM-DA incubated with 0.6 nM CaMKII peptide before being spin-coated on the glass cover slide surface and covered with physiological buffer. Error bars are the standard error in the mean of 15 experiments. (B) Single-molecule images of spin-coated CaM-DA molecules with a drop of experimental buffer placed on top of the surface. (C) Spin-coated CaM-DA molecules covered with a drop of experimental buffer containing 60 nM CaMKII peptide. (D) Spin-coated CaM-DA incubated with 60 nM CaMKII peptide before being spin-coated on the glass cover slide surface and covered with a drop of experimental buffer.

onto the glass cover slide surface at a concentration where an average of three to five CaM-DA molecules was contained inside the focal volume of the objective. The surface coverage for these measurements was determined by diluting a solution of known concentration and determining the single-molecule density of those dilutions. The slide was placed on the microscope, covered with physiological buffer, and a 488-nm excitation beam (1 kW/ cm2) was focused on the top surface of the glass cover slide. The acceptor fluorescence emission from the immobilized CaM-DA molecules was collected and binned in 2-ms intervals. Several trials were performed in different regions of the cover slide to validate the uniformity of the CaM-DA coverage. From each area examined, the average acceptor intensity was determined for the first 1000-1500 data points (2-3 s) after exposure to the excitation beam. This average acceptor emission intensity is shown in Figure 2A. To probe the ability of surface-immobilized CaM-DA molecules to bind a peptide target, surface-immobilized CaM-DA molecules were covered with a physiological buffer containing a saturating amount (0.6 µM) of CaMKII peptide. As Figure 2A shows, the intensity of acceptor emission is nearly unchanged when the peptide target is added to the immobilized CaM-DA molecules. The lack of the dramatic fluorescence quenching that accompanies 3634

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

binding of the CaMKII peptide illustrates the inability of surfaceimmobilized CaM-DA to bind a peptide target. The small decrease in the acceptor intensity of the spin-coated CaM-DA in the presence of CaMKII peptide can be attributed to quenching of a small number of CaM-DA molecules that diffuse away from the surface, bind a CaMKII peptide, and return to the surface, or to a small fraction of CaM-DA molecules that remained active on the surface. To verify that immobilization on a glass surface is responsible for the loss of target-binding functionality, CaM-DA was incubated with a saturating amount (0.6 µM) of CaMKII peptide in solution prior to spin-coating. These molecules were immobilized by spincoating and covered with physiological buffer. As shown in Figure 2A, the intensity of acceptor fluorescence dramatically decreases when CaM-DA is incubated with CaMKII peptide prior to immobilization on the glass surface. The bulk spectrum of CaM-DA shows the emission of Texas Red is quenched by ∼75% in the presence of the CaMKII peptide (see Figure 1B). The 80% quenching of acceptor fluorescence observed from CaM-DA in the presence of the CaMKII peptide in the subensemble experiment is in good agreement with the data obtained in the bulk. To assay the activity of surface-immobilized CaM-DA molecules at the single-molecule level, a dilute solution of CaM-DA was spin-

coated on a glass surface. Single-molecule assays are useful when limited amounts of analyte are available. By using dilute solutions of CaM-DA, the surface coverage was reduced so that a single molecule is present in the focal volume. The cover slide was covered with the same physiological buffer as in the subensemble experiments. Figure 2B shows a typical 10 µm × 10 µm acceptor fluorescence emission image from immobilized CaM-DA. A typical 10 µm × 10 µm image obtained when the physiological buffer is replaced with the physiological buffer containing 0.6 µM CaMKII peptide is shown in Figure 2C. The physiological buffer containing 0.6 µM CaMKII peptide was allowed to incubate for 5 min, and its addition results in little acceptor fluorescence intensity change. This demonstrates that the lack of target-binding functionality of CaM-DA at the single-molecule level for CaM immobilized at the glass surface. In contrast, when CaM-DA is incubated with 0.6 µM CaMKII peptide prior to being spin-coated on a glass cover slide surface, a dramatic decrease in fluorescent molecules is observed (Figure 2D). As in the subensemble measurements, this result verifies that surface immobilization impairs the function of CaM-DA. MBP‚CaM-DA Molecules Immobilized Away from the Surface Are Functional. As an alternative to immobilization of CaM-DA on a surface, we have developed a fusion construct that permits CaM-DA to be immobilized in an agarose gel.25 In a previous report, we have demonstrated that agarose gel does not in itself sufficiently restrict the diffusion of CaM to permit singlemolecule measurements over a time scale long enough for singlemolecule binding assays. CaM labeled with a fluorescent probe rapidly diffuses through the agarose gel matrix and is not rendered translationally immobile by the gel matrix.25 In contrast, we have shown that the fusion construct, MBP‚CaM-DA, is readily immobilized in an agarose gel, retains biological activity, and is immobile over a period of several minutes or longer.25 The agarose gel allows protein function to be maintained in a suitable buffer while restricting the translational motion of the protein. Thus, assays can be designed to monitor binding interactions of CaM with target drugs or peptides. We have demonstrated the utility of this construct for both ensemble and single-molecule assays. For ensemble measurements, a target was chosen with a dissociation constant in the micromolar range for CaM binding. Then, single-molecule assays are demonstrated with the CaMKII peptide, which has a picomolar dissociation constant. In addition to its many biological targets, CaM also binds small drug molecules with a relatively high affinity. One such family of drugs is the tricyclic antidepressants. Previous experiments have demonstrated the use of CaM as a biosensor for this class of antipsychotic drugs.20,33-35 The exact binding stoichiometry of amitriptyline is not known; however, an apparent inhibition constant of 2-26 µM has been reported.36 The micromolar inhibition constant implies a weaker interaction with CaM than that of the peptide fragment of CaMKII. Thus, amitriptyline is a good candidate for bulk binding assays because its dissociation constant is likely on the order of the nanomolar concentrations of CaM that are amenable to measurements by conventional fluorescence emission spectroscopy. Amitriptyline has been previ(33) Dikici, E.; Deo, S. K.; Daunert, S. Anal. Chim. Acta 2003, 500, 237-245. (34) Schauer-Vukasinovic, V.; Cullen, L.; Daunert, S. J. Am. Chem. Soc. 1997, 119, 11102-11103. (35) Johnson, J. D.; Wittenauer, L. A. Biochem. J. 1983, 211, 473-479. (36) Reynolds, C. H.; Claxton, P. T. J. Biochem. Pharmacol. 1982, 31, 419-421.

Figure 3. Quenching of MBP‚CaM-DA fluorescence (excited at 488 nm) in solution (9) and in a 1.5% agarose gel matrix (O) by the CaM antagonist amitriptyline. The data were fit by eq 1, and the results of the fit are shown for the solution (solid line) and gel experiments (broken line). The error for Kd was obtained from the error surface with the F statistic corresponding to one standard deviation.

ously reported to quench the fluorescence emission of CaM when a single Cys residue is genetically engineered at residue 109 and labeled with the coumarin derivative, MDCC.20 We therefore anticipated that the fluorescence of MBP‚CaM-DA will be quenched upon the binding of amitriptyline. Therefore, the binding of the CaM antagonist amitriptyline to MBP‚CaM-DA can be used to confirm the similar functionality of MBP‚CaM-DA in solution and immobilized in bulk agarose gels. To determine the dissociation constant of amitriptyline, MBP‚ CaM-DA (100 nM) was immobilized in a 1.0% agarose gel matrix inside a glass cuvette. The agarose gel was prepared by diluting a 2.0% stock gel solution with the physiological buffer and MBP‚ CaM-DA to a final concentration of 100 nM and a final agarose concentration of 1.0%. The gel was allowed to form for 5 min at room temperature and was shielded from light. After an initial fluorescence emission spectrum was obtained, a small volume (10 µL) of a concentrated amitriptyline solution, prepared with physiological buffer, was placed on top of the gel. The binding of amitriptyline yields a protein-drug complex where the acceptor fluorescence is mostly quenched; therefore, to determine a dissociation constant, the change in fluorescence intensity of the acceptor fluorophore was monitored as amitriptyline was titrated into the MBP‚CaM-DA solution. By acquiring the fluorescence emission spectrum in 2-min intervals and monitoring the intensity of the emission from the acceptor fluorophore, it was determined that a 10-min incubation period was sufficient for the drug solution to diffuse through the gel and reach equilibrium with the MBP‚ CaM-DA. The decrease in the fluorescence emission intensity of the acceptor was plotted against the concentration of amitriptyline, and the resulting binding curve is shown in Figure 3 (O). The dissociation constant Kd was calculated from the total concentrations of amitriptyline and MBP‚CaM-DA, [A] and [B], respectively, and the change in the fluorescence intensity of the acceptor (∆F), which was fit to the equation

∆F )

∆Fmax ([A] + [B] + Kd 2[A]

x([A] + [B] + Kd)2 - 4[A][B])

(1)

where ∆Fmax is the maximum quenching obtained over the range Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

3635

of amitriptyline concentrations employed. The values of ∆Fmax, Kd, and [B] were allowed to vary independently in the fit as the concentration of CaM decreases during the titration. The error given is the error at one standard deviation, calculated by the support plane method using the residuals of the fit. The fit of the titration curve yields a dissociation constant of 9.6 ( 4.1 µM for MBP‚CaM-DA binding amitriptyline in the gel and is in good agreement with inhibition constants obtained in previous assays performed with CaM.36,37 Previous work has shown that CaM may bind multiple amitriptyline molecules, but the inhibition constants for the binding of the second, third, and subsequent ligands are orders of magnitude greater in concentration than those examined here.37 For verification of the binding constant measured for MBP‚ CaM-DA in the gel, the binding of amitriptyline to MBP‚CaM-DA was measured in solution by titrating a concentrated solution of amitriptyline into a solution of 100 nM MBP‚CaM-DA prepared with physiological buffer. At each point in the titration, the solution was allowed to incubate for 5 min, after which the fluorescence emission spectrum of MBP‚CaM-DA was acquired. The intensity of the fluorescence emission was adjusted for the dilution associated with the addition of the drug solution. The binding curve obtained by plotting the change in fluorescence intensity of the acceptor fluorophore against the concentration of amitriptyline is shown in Figure 3 (9). A fit of the change in fluorescence intensity to eq 1 yielded a dissociation constant of 9.9 ( 2.4 µM. This dissociation constant is nearly identical to that obtained with MBP‚CaM-DA in agarose gel, indicating that the agarose gel matrix does not interfere with the ability of MBP‚CaM-DA to bind amitriptyline. It should also be noted that quenching curves for the solution and gel experiments were not normalized; therefore, the magnitude of the quenching of MBP‚CaM-DA fluorescence is independent of the medium. This further supports the conclusion that immobilization in an agarose gel does not alter the fluorescent characteristics of the MBP‚CaM-DA. Given the bulk immobilization result demonstrating that the target-binding function of MBP‚CaM-DA toward amitriptyline is not impaired when immobilized in an agarose gel, we next wanted to evaluate functionality of MBP‚CaM-DA as a single-molecule reporter of peptide binding. We chose to use the CaMKII peptide because it is thought to bind CaM with a picomolar Kd; thus, the probability of binding at low CaMKII concentrations is increased.24,38 Since binding of the CaMKII peptide dramatically quenches the intensity of the acceptor fluorophore, singlemolecule images from the acceptor channel could be used to evaluate the binding of the CaMKII peptide by MBP‚CaM-DA. Agarose gels for the single-molecule assay were prepared using the method described above (see Materials and Methods). Several 20 µm × 20 µm images were acquired at a resolution of 256 × 256 pixels for CaMKII peptide concentrations ranging from 0 to 1000 pm, while the CaM concentration was held constant. About 40 images were collected from a minimum of four different regions of the gel to ensure a uniform distribution of MBP‚CaM-DA molecules across the gel. Images collected in the absence of peptide target and at a CaMKII peptide concentration of 500 pM are shown in Figure 4. The loss of acceptor intensity corresponds directly with the addition of the CaMKII peptide. (37) Levin, R. M.; Weiss, B. J. Pharm. Exp. Ther. 1979, 208, 454-459. (38) Colbran, R. J.; Fong, Y. L.; Schworer, C. M.; Soderling, T. R. J. Biol. Chem. 1988, 263, 18145-18151.

3636

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

The ability to detect single MBP‚CaM-DA molecules and their quenching upon target binding suggests that target binding to MBP‚CaM-DA can be quantified by simply counting single molecules. Images were analyzed as described above (see Materials and Methods) to determine the average number of fluorescent MBP‚CaM-DA molecules in each image. The average number of molecules counted in an image is plotted in Figure 5 as a function of CaMKII peptide concentration. To determine the dissociation constant for CaMKII peptide bound to MBP‚CaM-DA, the data were fit to the following modified form of eq 1,

M)

VNAV (-[A] + [B] - Kd 2

x([A] - [B] + Kd)2 + 4Kd [B])

(2)

where M is the number of molecules counted, NAV is Avogadro’s number, [A] and [B] are the total concentrations of CaMKII peptide and MBP‚CaM-DA, respectively, Kd is the dissociation constant, and V is the effective excitation volume of the scan (2.4 × 10-12 L; 20 µm × 20 µm × 6 µm). In these fits, the concentration of MBP‚CaM-DA in the agarose gel is not known precisely due to the binding of MBP‚CaM-DA to the surface of the cover slides; therefore, the effective concentration of MBP‚CaM-DA and the value of Kd were both varied when fitting to eq 2. The error reported is the standard error of the fit. The binding constant determined from this assay is 103 ( 35 pM. This result demonstrates that gel-immobilized MBP‚CaM-DA can be used in an ultrasensitive assay of target binding. Since analyte molecules such as small-molecule drugs or peptides or even proteins as large as CaM itself are typically not immobilized in the gel, they can be added to the gel and the target binding activity of MBP‚CaM-DA monitored. The fluorescent MBP-CaM construct offers the potential to develop assays to detect and characterize CaM interactions with peptides, proteins, or drugs. While specific only to CaM, the coupling of this assay to a microfluidic environment would allow the interaction of CaM with its many protein targets to be studied with greatly increased throughput. Whereas other screening techniques such as two-hybrid screening assays can probe protein-protein interactions with very high throughput, it requires construction of fusion proteins with both interaction partners and provides limited quantitative information regarding the binding strength of the interaction.39-41 Surface plasmon resonance (e.g., Biacore) analyzes protein-protein interactions by immobilizing the analyte very close to the surface to allow excitation by total internal reflectance; this technique is therefore susceptible to the same experimental artifacts as the aforementioned single-molecule experiments performed at or near a surface.42 Fluorescence correlation spectroscopy can provide sensitive information regarding protein-protein interactions; however, this technique is limited by the short, diffusion-limited residence time of the molecule in the focal beam and requires sophisticated liquid-handling devices to be reusable.43,44 (39) Jaeger, S.; Brand, L.; Eggeling, C. Curr. Pharm. Biotech. 2003, 4, 463476. (40) Fashena, S. J.; Serebriiskii, I.; Golemis, E. A. Gene 2000, 250, 1-14. (41) Uetz, P.; Hughes, R. E. Curr. Opin. Microbiol. 2000, 3, 303-308. (42) Hall, D. Anal. Biochem. 2001, 288, 109-125.

Figure 4. Representative 20 µm × 20 µm images of the acceptor fluorescence of MBP‚CaM-DA immobilized in a 1.5% agarose gel (A) in the absence and (B) in the presence of 500 pM CaMKII peptide. Excitation was at 488 nm.

Figure 5. Number of molecules observed in the 20 µm × 20 µm images of the acceptor fluorescence of MBP‚CaM-DA (excited at 488 nm) shown as a function of the CaMKII peptide concentration. The error bars represent the standard deviation of the number of molecules observed in ∼40 scans. The solid line is the fit of the data to eq 2, which yields a dissociation constant of 103 ( 35 pM.

CONCLUSIONS We have demonstrated the use of the fusion protein MBP‚ CaM-DA for assays of CaM-target binding interactions. This energy-transfer fusion construct is easily immobilized in a low weight percent agarose gel and moreover has been shown here to exhibit the same affinity for the CaM antagonist amitriptyline when immobilized in the agarose gel as in solution. In contrast, we have shown that surface-immobilized molecules do not retain their ability to bind a peptide target, the CaM binding domain of CaMKII. Because the agarose concentration is low, the immobilized molecule is surrounded by an environment composed (43) Koltermann, A.; Kettling, U.; Bieschke, J.; Winkler, T.; Eigen, M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1421-1426. (44) Auer, M.; Moore, K. J.; Meyer-Almes, F. J.; Guenther, R.; Pope, A. J.; Stoeckli, K. A. Drug Discovery Today 1998, 3, 457-465.

of nearly 99% physiological buffer. Additionally, the potential of such immobilization to be coupled with methods that allow many assays to be performed simultaneously would increase the throughput of single-molecule assays using immobilized molecules. Using an assay where the MBP‚CaM-DA energy-transfer construct reports the binding of a peptide target by a dramatic decrease in fluorescence intensity, we detected binding of the CaMKII peptide with the construct. Single-molecule assays of CaM binding a peptide fragment of CaMKII demonstrate that the targetbinding functionality of MBP‚CaM-DA is retained when it is immobilized in agarose gels. The binding strength of MBP‚CaMDA to the CaMKII peptide was determined by counting singlemolecule fluorescence images. A dissociation constant of 103 ( 35 pM was determined by this method. This article presents a novel method to investigate target-binding equilibrium at the single level and suggests the possibility to interrogate atto- and zeptomolar concentrations directly by the counting of individual binding events. ACKNOWLEDGMENT We thank Kenneth Osborn for development of the software for data acquisition and analysis, and Jay Unruh for his assistance with the preparation of the titration curves and fitting. Funding was provided from the National Institute of Health (R01 GM58715), the American Heart Association (99513262), and a Research Corporation Research Opportunity Award. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review February 11, 2004. Accepted March 25, 2004. AC0497656

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

3637