THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1964 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 9. Issue of May 10, pp. 5717-5725,19&4 Printed in U.S.A.

Fluorescence Energy Transfer between Cobra a-Toxin Molecules Bound to the Acetylcholine Receptor*

(Received for publication, August 22, 1983)

David A. Johnson$, Judith G. Voetj, and Palmer Taylor From the Division of Phurmncobm. “013 H, Department of Medicine, University of California, Sun Diego, La Jolla, California 92093

” . . -

An approach was developed with steady state fluo- rescence energy transfer measurements to examine the spatial relationship between the two a-toxins bound to the acetylcholine receptor. By taking advantage of the slow dissociation rates of a-toxins (Naja naja siamen- sis 3) from the receptor and of the equal probability with which a-toxins bind to the two a-toxin-binding sites, we derived an equation which allows prediction of a “true” efficiency of transfer based on the relation- ship between fractional site occupancy and the ob- served transfer efficiency ascertained from donor quenching. Using this approach, we examined the ef- ficiency of energy transfer between two fluorescently labeled a-toxins, W-fluorescein isothiocyanate lysine 23 a-toxin and monolabeled tetramethylrhodamine is- othiocyanate a-toxin bound to the receptor from the Torpedo californica electric organ. Significantly greater (32 versus 14%) energy transfer was observed with the membrane-associated than with the solubi- lized receptor, suggesting that transfer between fluo- rophores on separate receptor molecules is greater than that occurring intramolecularly between the two sites on the receptor. The magnitude of the distances calculated from the intrareceptor energy transfer ef- ficiency combined with the considerable inter-receptor energy transfer indicate that the fluorophores would reside on the outer perimeter of the receptor molecule rather than near the central axis perpendicular to the plane of the membrane.

The AChR’ is a cation channel whose activation is elicited by acetylcholine. Owing to its relative abundance, the most thoroughly characterized AChR comes from the Torpedo cal- ifornica electric organ (1-3). In receptor-rich membrane frag- ments from the Torpedo, the AChR exists as tightly packed, disc-shaped particles of 90 f 10 A in diameter, with a central 15-20-A cavity (4-6). The pentameric receptor is comprised of four different subunits, designated as az, p, y, and 6 (7,8).

* This work was supported in part by United States Health Service Grants GM 18360 and 24437. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be sent. J Recipient of the Eugene Lang Swarthmore Faculty Fellowship.

Present address, Department of Chemistry, Swarthmore College, Swarthmore, PA 19081,

The abbreviations used are: AChR, nicotinic acetylcholine recep- tor; FITC-toxin, N’-fluorescein isothiocyanate lysine 23 a-toxin; TRITC-toxin, tetramethylrhodamine isothiocyanate a-toxin; a- toxin, Naja naja siamensis 3 a-cobra toxin, DANSYL, 5-dimethyla- mino-naphthalene-1-sulfonyl; kl, association rate constant; k l , dis- sociation rate constant.

The a-subunits contain the primary binding site for acetyl- choline and constitute the major surface with which the a- toxins associate. The five subunits are organized like barrel staves about the central cavity which is thought to be the ion channel (11, 12). Based on an electron microscopic examina- tion of detergent-solubilized avidin-biotinyl-a-toxin-AChR complexes, Karlin and his associates (13-15) concluded that the arrangement of the subunits around the perimeter of the central cavity is aya6p or ayaP6. Using electron microscopy and single-particle image averaging with 20-A resolution, Zingsheim et al. (16) showed that a-bungarotoxin binds to two regions of the receptor: one adjacent to the &subunit and the other diametrically across the molecule, -50 A away from the &subunit.

Since fluorescence resonance energy transfer techniques have the capacity to measure distance between chromophores on a functional molecule and obviate the use of fixatives or frozen preparations, we developed a framework to study the spatial relationship between a-toxin sites on the AChR by this technique. To this end, we examined the efficiency of energy transfer between two fluorescent-labeled a-toxins, Iy- sine 23 FITC-toxin and monolabeled TRITC-toxin, bound to membrane-associated and detergent-solubilized Torpedo AChR. a-Toxins bind randomly to the two primary sites on the receptor. To account for the population of donor-acceptor pairs within the total population of toxin-receptor complexes, we derived an equation that defines the relationship between donor occupation and energy transfer.

EXPERIMENTAL PROCEDURES

Mater&-Fluorescein and tetramethylrhodamine (isomer R) were obtained from Polysciences and Baltimore Biological Labora- tories, respectively. Cobra a-toxin (siamensis 3 ) was isolated following the method of Karlsson et al. (17) from the venom of Naja m j a siamensis; venom was obtained lyophilized from Miami Serpentarium. FITC-toxin was prepared as described elsewhere (18) and subse- quently purified by column isoelectric focusing as described previously (19). Analytical isoelectric focusing of this material indicated >98% homogeneity of fluorescent material. Na’%I was purchased from New England Nuclear. Mono-[’261]iodotyr~sine 25 a-toxin (siamensis 3 ) was prepared and separated from noniodinated and diiodo species by column isoelectric focusing (19). All other reagents were at least reagent grade.

TRZTC Labeling of a-Toxin-Freshly thawed cobra a-toxin (3.69 pmol) in 3.0 ml of 100 mM sodium phosphate buffer, pH 7.0, was mixed with 7.38 pmol of TRITC (3.27 mg/ml in 0.4 M NaHC03) and the pH was adjusted to 9.1 with 1.0 M NaOH. This mixture was allowed to react for 30 min under Nz at room temperature with stirring. Crystalline DL-lysine (20 mg) was added to quench the reaction for 5 min. The reaction mixture was then passed through a Bio-Gel P-6 column (2.5 X 25 cm) equilibrated with 0.03 M ammonium acetate, pH 4.8 (buffer I). Void volume fractions were pooled and applied to a carboxymethylcellulose (Whatman CM52) column (0.9 X 100 cm) at 4 “C. The column was washed with 50 ml of buffer I, and the various peptides eluted with a linear gradient formed from 1

5717

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5718 Energy Transfer between a-Toxin-binding Sites liter of buffer I and 1 liter of 0.14 M ammonium acetate. The major fluorescent peaks were pooled, lyophilized, resuspended in 0.1 M ammonium acetate, and stored at -70 “C.

Receptor Isolation-Receptor-rich membrane fragments were iso- lated from T. californica electric organ following published procedures (18, 20). Aprotin (2.7 trypsin inhibitor units/mg/100 g of tissue) was also added during the initial homogenization. The specific binding activities of the receptor preparations were measured by adsorption of ‘“I-labeled a-toxin-receptor complexes to DEAE-cellulose filters (21) and ranged from 1 to 2 nmol of a-toxin binding sites/mg of protein. Incubation of a 10-fold excess of native a-toxin 30 min prior to the addition of ‘251-a-toxin defined the “nonspecific” binding component in the total binding.

Electrophoresis and Isoelectric Focusing-Sodium dodecyl sulfate- polyacrylamide gel electrophoresis methods combined a discontinuous buffer system (22) with the gel porosity formulations of Weber et al. (23). To the top of the 18% acrylamide separating gel, a 3.3% stacking gel was layered. All samples were reduced with 1% mercaptoethanol prior to electrophoresis. The electrode buffer was 25 mM Tris, 0.19 M glycine, pH 8.3, 0.1% sodium dodecyl sulfate. Column isoelectric focusing was performed as previously described (19).

Spectroscopy-With the exception of kinetic experiments, all flu- orescence measurements were made on a Farrand Mark I spectro- fluorometer equipped with corrected excitation and a Hamamatsu R928 photomultiplier tube. The spectrofluorometer was interfaced to a Tektronix 4052 microcomputer with a real-time clock via a TransEra analog-to-digital converter. For spectral scanning, the an- alog-to-digital converter averaged 324 samples/s/nm and for fixed wavelength determinations averaged 15,000 samples/point. The spec- tral sensitivity of the emission monochromator and photomultiplier system was determined using a BaS04 pellet (Eastman Reflectance Kit) in the sample compartment, setting both excitation and emission monochromators at the same wavelength and measuring photomul- tiplier current at 10-nm intervals with the instrument in the corrected excitation mode. From this spectral sensitivity profile, emission cor- rection coefficients were generated to correct emission spectra and stored into the computer. Except where noted, all spectra were taken with the samples in 100 mM NaCl, 10 mM sodium phosphate at pH 7.4. The quantum yield of the FITC-toxin was determined by com- parison of the integrated areas under the corrected emission spectra for solutions of FITC-toxin in buffer and of fluorescein in 0.1 N NaOH ( Q = 0.85 (24)) of equal absorbance at 450 nm.

Absorption spectra were measured with a Cary 16 spectropho- tometer interfaced to the Tektronix microcomputer.

Binding Kinetics of the Labeled Toxins-See Miniprint? Energy Transfer-The efficiency, E, of dipolar resonance energy

transfer between a discrete donor and acceptor pair is related to the distance ( R ) separating the pair by the following equation (28, 29):

R = Ro(l/E - 1)”‘j (9)

Ro, the distance at which transfer efficiency equals 50%, equals

9.765 X 103(p.J .Q~.n“)’ /6 (10)

The overlap integral, J , represents the degree of resonance between excited state donor and acceptor dipoles and is evaluated as the integrated mutual area of overlap between the donor emission spec- trum, ID(X), and the acceptor absorption spectrum, CA(X).

I ~ ( x ) c ~ ( x ) x ‘ d X

J’ ID(X)dh J = (11)

QD denotes the donor quantum yield in the absence of acceptor and n the refractive index of the medium between donor and acceptor. K2, the “orientation factor,” accounts for the relative orientation of

’ Portions of this paper (including “Experimental Procedures,” “Results,” Figs. 3-6, and an Appendix) are presented in Miniprint at the end of this paper. Miniprint is easily read with the aid of a

the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, standard magnifying glass. Full size photocopies are available from

MD 20814. Request Document No. 83M-2422, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

the donor emission and acceptor absorption transition dipoles. While it is usually not possible to directly measure P, upper and lower bounds were obtained by measurement of the rotational mobility of the donor and acceptor. When both axial depolarization factors are positive, the upper and lower limits of the orientation factor can be calculated from the following equations:

Kkm = ‘h(1 + (dD ’ ) + ( d ~ ‘ ) +3(d~’)(d,4~)) (12)

K L = - ( ( d ~ ’ ) (dA”))/2] (13)

where ( L I D ” ) and (d.,‘) respectively denote the axial depolarization factors for donor and acceptor (30).

These axial depolarization factors were estimated from the individ- ual emission anisotropies of each labeled toxin free in solution mea- sured at various viscosities (v). From a plot of reciprocal anisotropy uersus temperature divided by viscosity (T/v), the intrinsic anisotropy was obtained from the intercept at infinite viscosity. Division of each intrinsic anisotropy by 0.4 yielded the estimate for the axial depolar- ization factor (30).

In the typical case where donor and acceptor are at separate sites on a macromolecule, the efficiency of energy transfer (ED) can be measured as the extent of reduction of donor quantum yield.

where IDA and I D are the fluorescence intensities in the presence and absence of acceptor, respectively.

The efficiency of energy transfer can also be measured as the extent of acceptor-sensitized emission upon excitation at wavelengths where donor absorbance predominates using the following equation:

(15)

The function fDA(XD, X) is the scatter-corrected spectrum in the region of acceptor emission of the sample with both donor and acceptor present, and the functions fD(hD, h) andfa(XD, X) are scatter-corrected spectra of samples, respectively, with only donor or only acceptor present. AD(XD)/AA(XD) represents the ratio of absorbances of donor and acceptor at the excitation wavelength (31).

In the special case where donor and acceptor bind nonpreferentially to either of two sites on a macromolecule, the “true” transfer effi- ciency can be estimated from a knowledge of the observed transfer efficiency monitored by donor quenching, ED&, the fractional occu- pancy by donors, y, the donor dissociation constant, KD, the concen- tration of binding sites, [R],, and the change of donor fluorescence upon binding, Qb/Qr by the following equation (cf. Appendix).

EA& = fDA(xD, X) - fD(XD, X) - f A ( b

A D ( ~ ) / A A ( X D ) . ~ A ( ~ D ~ x) - f D ( b X)

Receptor Occupancy-Since the rate of ‘=I-a-toxin binding is pro- portional to the concentration of unoccupied sites (19,25), measure- ment of the initial apparent association rate constant of ‘%I-cY-toxin to the AChR assesses a-toxin occupancy of receptor sites. Incremental quantities of either FITC-toxin or TRITC-toxin were incubated with the AChR for 2 h prior to the addition of ‘%I-a-toxin, except where noted. Aliquots were taken at 20, 40, and 60 s and spotted on DE81 filters which were then washed twice for 15 min in 10 mM sodium phosphate buffer, pH 7.4, containing 0.1% Triton X-100. Data were plotted to the integrated rate expression for a bimolecular reaction. The fractional binding rates were used to assess the fraction of unoccupied a-toxin sites.

RESULTS

Preparation of TRITC-labeled a-Toxin (TRITC-toxin)- Cobra a-toxin was reacted with a 2-fold excess of TRITC as described under “Experimental Procedures.” Elution profiles of fluorescence and protein after purification by ionic strength gradient elution from a CM52 column are shown in Fig. 1. Most of the conjugated a-toxin eluted as what appeared to be two incompletely resolved peaks eluting just before native a- toxin. The integrated areas showed that about 30% of the native a-toxin was conjugated. In the present study, only the

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Energy Transfer between a-Toxin-binding Sites 5719

W 0

20 40 60 80 100 120 140 160 IS0

FRACTION NUMBER

FIG. 1. Elution profile of TRITC-a-cobra toxin reaction mixture following CM52 ion exchange chromatography. C - *, relative fluorescence; o " 0 , protein; - - -, conductivity of effluent. Conjugated a-toxin (3.69 pmol) was applied to a 0.9 X 100 cm column. After washing with 50 ml of buffer, elution was accom- plished with a 0.03-0.14 M ammonium acetate gradient. The fraction size was 8.2 ml. Horizontal bar above second major fluorescent peak indicates fractions pooled and designated TRITC-toxin. The protein concentration was determined by the method of Lowry et al. (38) with reference to bovine serum albumin.

major fractions associated with the peak of conjugated a- toxin nearest the native a-toxin peak were utilized. The conjugated a-toxin sample was lyophilized and passed over a Bio-Gel P-10 column to remove traces of unconjugated TRITC which remained. Following sodium dodecyl sulfate electrophoresis of an aliquot of this material (0.35 nmol), no unconjugated TRITC fluorescence could be detected. Upon column isoelectric focusing of the TRITC-toxin, all detectable fluorescence focused as a single, but somewhat broad, peak at PI = 9.9 (data not shown). Under the same conditions, native toxin focuses with a PI of 10.7 (19).

Delineation of the site(s) of labeling of TRITC proved impossible since, after reaction of free TRITC with H20, several 550-nm absorbing peaks were partially separated on HPLC. This heterogeneity in the TRITC itself was the likely cause of our inability to purify labeled peptides.

Spectroscopic Characterization-Absorption and corrected emission spectra of FITC-toxin and TRITC-toxin are shown in Fig. 2 and the spectroscopic constants are summarized in Table I. The quantum yield of the FITC-toxin free in the buffer was 0.165. Since the binding of FITC-toxin to the membrane-associated AChR is associated with a 2.05-fold enhancement of fluorescence (25), the quantum yield of FITC- toxin in the bound state is therefore 0.34. Solubilization of the FITC-toxin-AChR complex by the addition of 2% sodium cholate has no effect on the emission or excitation spectra.

An absorbance maximum of the TRITC-toxin was found at 547 nm and yielded an extinction coefficient of 72,000 M" cm" in buffer. This value is similar to a value of 84,000 M" cm" that we observed for TRITC in the same buffer, sup- porting the electrophoretic evidence that TRITC-toxin is a monoconjugate. The corrected emission maximum of the TRITC-toxin was found to be 576 nm. Upon binding to the membrane-associated AChR, its fluorescence increases 3.8- fold without a detectable shift in the maximum wavelength. This behavior differs from the other major TRITC-conjugate that eluted before TRITC-toxin (cf. Fig. 1). Upon binding to the membrane-associated AChR the other major TRITC- conjugate present in the neighboring peak exhibits only a 1.2- fold increase in quantum yield.

Kinetics of TRITC-toxin Binding-Table I1 summarizes the kinetic constants derived from the slopes of plots of integrated rate equations (Equations 3 and 4). Within a factor of 2, the

80

WAVELENGTH (nm) FIG. 2. Spectral relationship between donor-FITC-toxin

and acceptor-TRITC-toxin energy transfer pair. The absorp-

extinction coefficients (M-' cm"). The corrected emission spectra of tion spectra of donor (-1 and acceptor (- . --) are presented as

donor ( . . . . .) and acceptor (---I bound to the AChR are presented in arbitrary intensity units and are not shown in proportion to the relative quantum yield. All spectra were measured in 100 mM NaC1, 10 mM sodium phosphate a t pH 7.4.

TABLE I

FZTC-toxin and acceptor-TRZTC-toxin on membrane-associated and Energy transfer parameters and spectroscopic constants for donor-

solubilized AChR Energy transfer parameters

Energy transfer efficiency RZI3'

A A

Membrane-associated 0.32' 46-70 56 Solubilized 0.14' 55-84 67

AChR

Spectroscopic constants

Q D ~ J' K1' R08

cme/mol A 0.34 2.9 X 1013 0.21-2.5 40.7-61.7

Distance calculated by employing K2 = 2/3.

bFrom an interpolation to zero FITC-toxin in the plot of the specific energy transfer in membrane-associated AChR as a function of donor occupancy (bottom of Fig. 9).

E From the best fit (Fig. 10) of the donor-occupancy dependence of apparent transfer efficiency to the two-equivalent-site model (Equa- tion 16).

NaOH (24). Quantum yield based on a value of 0.85 for fluorescein in 0.1 N

e Calculated from Equation 11. 'Calculated from Equations 12 and 13. 8 Calculated from Equation 10 using measured values for K$,, and PmX and assuming a refractive index of 1.4.

rate constants derived from total fluorescence and polariza- tion agree, and hence do not reveal kinetic heterogeneity in the TRITC-toxin sample. The association rate constant is about 10 times slower, and dissociation rate constant is about 3 times faster for the solubilized compared to the membrane- associated AChR, therefore, the dissociation constant esti- mated from the ratio k-,/kl is about 30 times larger for the solubilized AChR. It is unclear why cholate dramatically decreases the affinity of the TRITC-toxin toward the AChR. Solubilization alone cannot account for this effect, because Triton X-100 solubilization does not alter the binding kinetics of m~no['~~I]iodotyrosine 25 cobra a-toxin toward the AChR (19).

Kinetics of FITC-toxin Binding to the Solubilized AChR-

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5720 Energy Transfer between a-Toxin-binding Sites TABLE I1

TRITC-toxin rate constants of association to (k,) and dissociation from fk-1) the membrane-associated and cholate-solubilized AChR Constant values are based on the least squares fit to the left-hand

portion of the integrated rate equations for a reversible bimolecular reaction (Equation 3) or unimolecular dissociation (Equation 4). Numbers in parentheses, number of determinations.

Membrane-associated Solubilized

AChR fluorescence data k, ("I s-') 54 f 11 X lo3 (6) 7.5 f 1.3 X lo3 (6) k-, (s-') 4.2 f 0.5 X lo-' (2) 15 f 5 X lo-' (2)

k-,lk, (nM) 0.8 20

kl (M-' s-') 81 f 2 X IO3 (6) 7.0 f 1.7 X lo3 (6) k-I (s-') 4.8 f 0.5 X (2) 16 f 1 X (2)

t1/2 (hIb 4.6 1.3

AChR polarization data

t1/2 (hY 4.0 1.2 k-llk, (nM) 0.6 23

"Dissociation half-time, t1/2 = In 2/k-,.

We have previously described the kinetics of FITC-toxin binding to the membrane-associated AChR. Based on the integrated rate analyses described for the TRITC-toxin, the association rate constant of FITC-toxin is decreased slightly from 6.7 k 1.7 X lo3 (25) M-' s" to 4.1 f 0.7 X lo3 (seven determinations) upon solubilization of the AChR. Similarly, the dissociation constant increased from 3.3 x s-l (25) (tIl2 = 5.8 h) to 11 f 5 X s-' (tllz = 1.8 h) (five determinations) so that the dissociation constant estimated from the ratio of the rate constants is about 5 times higher (27 compared to 5 nM).

Energy Transfer in the Membrane-associated AChR Esti- mated with 50% Donor and Acceptor Occupancy-To evaluate energy transfer between AChR a-toxin sites, quantities of FITC-toxin and TRITC-toxin sufficient to occupy 50% of the '251-a-toxin sites within 1 h were initially determined. The quantity of FITC-toxin that yielded 50% site occupation was incubated with the membrane-associated AChR for 1 h fol- lowed by a 30-min incubation with the quantity of TRITC- toxin necessary to approach saturation of the remaining sites. To confirm our observations with a reversed order of addition, the same quantity of TRITC-toxin was incubated with mem- brane-associated AChR in a separate cuvette for 30 min followed by a 1-h incubation with the amount of FITC-toxin necessary to occupy the remaining sites. The emission spectra of the above samples, along with spectra of control samples where no energy transfer is present, are shown in Fig. 7. With excitation at 450 nm, little or no detectable TRITC-toxin emission is observed near the peak of FITC-toxin emission, demonstrating that the observed decrease in the FITC-toxin emission spectra in the presence of TRITC-toxin, compared to the sum of control spectra, represents donor quenching. Further support for occurrence of energy transfer between a- toxin sites stems from the observed enhancement of TRITC- toxin emission at -576 nm from the sample where both FITC- toxin and TRITC-toxin are bound to the AChR. With 50% FITC- and TRITC-toxin occupancy, the average (fS.D.) ap- parent donor quenching (Equation 14) and acceptor sensiti- zation (Equation 15) were 0.29 k 0.05 and 0.19 f 0.07, respectively (six determinations).

Donor Occupancy Dependence of Resonance Energy Trans- fer in Membrane-associated AChR-To further quantify res- onance energy transfer between a-toxin sites of the mem- brane-associated AChR, the dependence of site occupancy of the donor on fractional quenching was examined by titrating TRITC-toxin into preparations with 15 and 75% of the toxin sites occupied with FITC-toxin. The site specificity of the

500 600

Wavelength (nn)

FIG. 7. Corrected emission spectra of FITC-toxin and TRITC-toxin each bound to 60% of the membrane-associated AChR toxin-binding sites. Top, dashed line, AChR (-120 nM in toxin sites) was incubated at room temperature for 1 h with sufficient FITC-toxin (65 nM) to occupy 50% of the sites, and then sufficient TRITC-toxin (65 nM) to occupy 50% of the sites was added and the mixture was incubated an additional 0.5 h; solid line, sum of inde- pendently determined spectra of the above amounts of FITC-toxin and AChR after 1-h incubation and of TRITC-toxin and AChR after 30-min incubation. Bottom, same as top except that TRITC-toxin was incubated with the AChR before the addition of FITC-toxin. All spectra were corrected for light scatter (excitation at 450 nm with a Farrand 450 filter) and measurements were made in 100 mM NaC1, 10 mM sodium phosphate at 7.4.

transfer process was also assessed. In separate cuvettes after the FITC-toxin was incubated with AChR as above, excess native toxin was added prior to the titration of TRITC-toxin to prevent the specific binding of the TRITC-toxin. The apparent donor quenching as a function of TRITC-toxin added is shown in Fig. 8. As would be expected with higher donor occupancy and concomitant occupancy of both sites on each AChR molecule by donor-labeled toxins, less total trans- fer was observed at higher FITC-toxin occupancies. Nonspe- cific donor quenching was much less dependent on donor occupancy and approached a linear function of TRITC-toxin concentration. An apparent site-specific transfer efficiency was estimated by subtracting the nonspecific from total trans- fer efficiency and is represented in Fig. 8 as a dashed line. The specific transfer efficiency appears to saturate at quan- tities below 250 nM TRITC-toxin. The dependence of this maximal transfer efficiency on FITC-toxin occupancy was next determined.

To reduce the exposure time of the TRITC-toxin to the FITC-toxin-AChR complexes and thereby minimize the dis- placement of FITC-toxin by the TRITC-toxin, only a single saturating concentration (250 nM) of TRITC-toxin was added to a series of cuvettes with incremental differences in FITC- toxin occupancy. The results of this experiment are presented at the top of Fig. 9. As in the previous experiment, the apparent total transfer efficiency was inversely related to FITC-toxin occupancy and the nonspecific transfer efficiency was not affected appreciably by fractional FITC-toxin OCCU- pancy. The apparent specific transfer efficiency is shown as a function of FITC-toxin occupancy at the bottom of Fig. 9. Interpolation of this plot to zero FITC-toxin occupancy

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Energy Transfer between a-Toxin-binding Sites 5721

" _ " 0-" """ """"_" 6

0 , I

I I

W

0.4

0.3 c

0.2 """__"_ _""""""""""~

100 300 500 TRITCeToxin (nM)

FIG. 8. Apparent energy transfer efficiency between FITC- toxin and TRITC-toxin bound to the membrane-associated AChR (100 nM in toxin sites) determined by donor quenching as a function of the concentration of TRITC-toxin. AChR was incubated for 1.5 h with sufficient FITC-toxin to occupy 15% (top) or 75% (bottom) of the sites before native toxin (300 nM) was incubated for an additional 30 min in one set of paired cuvettes to prevent specific TRITC-toxin binding. At 10-min intervals, incre- mental amounts of TRITC-toxin were then added and emission recorded at the FITC peak emission wavelength (excitation at 450 nm, emission at 519 nm with a Corning 3-70 filter). The apparent transfer efficiency was calculated using Equation 14 after subtraction of the scatter blank. B, total transfer efficiencies measured in the absence of native toxin; 0, apparent nonspecific transfer efficiencies in the presence of excess native toxin to prevent specific TRITC- toxin binding; 0, difference between the total and nonspecific transfer efficiencies.

yielded an apparent specific transfer efficiency of 32%. Along with the specific transfer efficiencies are theoretical plots of apparent quenching transfer efficiency versus donor site oc- cupancy for various transfer efficiencies based on a model of two equivalent sites for toxin binding (Equation 16). Although the apparent specific transfer efficiencies decrease with FITC- toxin occupancy, the data points are poorly fit by the model.

Resonance Energy Transfer in Solubilized AChR-Since the occupancy dependence of the apparent specific transfer effi- ciency in the membrane-associated AChR did not fit the model for two equivalent sites (Equation 16), energy transfer between more than a single pair of toxin sites presumably was occurring. Total energy transfer might be expected to include transfer between receptor molecules in the densely packed membranes in addition to the intramolecular transfer pre- dicted by the two-site model. To eliminate potential transfer between toxin sites on separate AChR molecules, donor quenching was measured after solubilization of the receptor- toxin complexes with 2% sodium cholate. The same protocol utilized for Fig. 9 was performed except that 20 min after the TRITC-toxin was added the membranes were solubilized with 2% cholate for 1 min before the FITC-toxin emission was recorded. The results of this experiment are shown in Fig. 10. Both the total and specific transfer efficiencies were signifi- cantly less than observed for the membrane-associated AChR (Fig. 9). Although transfer was reduced, the apparent specific donor quenching data now fit the two-equivalent-site model

0-4.

0.3.

0.4 D -TX

0 0 o +TX

I

0.5

Fractional FITC-Toxin OcCuPancY FIG. 9. Donor occupancy dependence of observed quenching

transfer efficiency between FITC-toxin and TRITC-toxin bound to the membrane-associated AChR. AChR (100 nM in toxin sites) was incubated for 1.5 h with various amounts of FITC- toxin. Native toxin (300 nM) was incubated for an additional 30 min in one set of cuvettes to prevent the "specific" binding of TRITC- toxin. TRITC-toxin (250 nM) was then added to both sets of cuvettes and incubated for 20 min before the peak emission (excitation at 450 nm, emission at 519 nm) of the FITC-toxin was measured. The apparent transfer efficiency was calculated using Equation 14 after subtraction of scatter blank. Top, represent apparent total transfer efficiency in absence of native toxin; 0 represent apparent nonspecific transfer efficiencies in the presence of excess native toxin to prevent TRITC-toxin binding. Bottom, represent apparent specific transfer efficiencies, i.e. the difference between total and nonspecific transfer efficiencies. The solid lines show a theoretical plot of the functional relationship between fractional donor occupancy and observed trans- fer efficiency for indicated levels of true transfer efficiencies utilizing Equation 16 and values for donor K d , Qb/Q,, and [ R ] , of 5 nM, 2.05, and 100 nM, respectively.

(Equation 16) reasonably well with a true transfer efficiency of 14%.

DISCUSSION

To demonstrate the utility of dipolar resonance energy transfer to examine the spatial relationship between ligands bound to the AChR, we prepared a site-specific FITC-labeled toxin conjugate and a mono-TRITC-labeled toxin. The ra- tionale behind selection of this pair of ligands for our initial studies involved several considerations. First, the presence of two a-toxin-binding sites per receptor oligomer seems well established. Although conjugation with the fluorophores re- duces the affinity of the toxins, their dissociation rates are sufficiently slow (cf Table 11) to complete energy transfer measurements without appreciable exchange of a-toxin mol- ecules between sites. Second, both FITC-toxin and TRITC- toxins are fluorescent. Therefore, dipolar resonance energy transfer can be established as the mechanism of donor quenching by the near equivalence of donor quenching (29 5%) and acceptor sensitization (19 2 7%) at 50% donor and acceptor occupation. Since donor quenching can occur through mechanisms other than dipolar energy transfer par- ticularly in macromolecules where conformational changes

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5722 Energy Transfer between a-Toxin-binding Sites

0.3.

I W Y

0.3. I c

1

0.5 1.0

Fractional FllC-Toxin Occupancy

FIG. 10. Donor occupancy dependence of observed quench- ing transfer efficiency between FITC-toxin (TX) and TRITC- toxin bound to the cholate-solubilized AChR. The description of this experiment is exactly the same as described in Fig. 9 except that 1 min before the peak FITC-toxin emission was measured (excitation at 450 nm, emission at 519 nm with a Corning 3-70 filter), each sample was solubilized in 2% cholate.

might be anticipated, it becomes important for estimates of intersite distance to establish that dipolar resonance energy transfer is the predominant mechanism of quenching. Accep- tor sensitization is inherently a less accurate measurement, since donor and acceptor absorbances are included in its estimation and there are uncertainties in determining wave- length bandwidths. The demonstration that FITC-toxin is labeled on lysine 23 and the kinetics of its interaction with receptor-containing membranes have been described else- where (17, 25). Isolation and identification of a single site- specific TRITC-conjugate using conventional chromato- graphic procedures proved more intractable. The major rea- sons for our difficulties were low purity of commercially available TRITC and the relatively small difference between isoelectric points of TRITC-toxin conjugates. In spite of this limitation, we have isolated a fraction of TRITC-labeled toxin which shows isoelectric homogeneity and relative consistency between binding rate constants as derived from the total fluorescence and polarization data. The isoelectric profile indicates that only one lysine has been labeled but we cannot be sure of the microscopic homogeneity of labeling at a single residue in the linear sequence.

With this sample of TRITC-labeled toxin and the FITC- toxin, we measured the apparent efficiency of energy transfer between the fluorophores bound to the membrane-associated and solubilized Torpedo AChR. Interestingly, energy transfer is far greater (32 uersus 14%) in the membrane-associated receptor (Table I), which suggests that significant intermolec- ular transfer occurs in addition to the expected intramolecular transfer between the two a-toxin sites within the receptor oligomer. Several lines of evidence support this suggestion. Since membrane-associated AChRs are densely packed with estimates of up to 20,000 a-toxin sites/pm2 (1-3), the distance between a-toxins on adjacent AChRs would be small if toxins

bind near the perimeter of the AChR. Moreover, analysis of the packing arrangements and nearest neighbors of the recep- tor indicates that more than a single acceptor could be in proximity to each FITC donor. Similarly at high donor occu- pancies, multiple donors could transfer energy to a single acceptor. This is, in fact, consistent with the nonlinear de- pendence of transfer efficiency on donor occupancy (Fig. 9). Cholate has no detectable effect on the excitation or emission spectra of either FITC-toxin or TRITC-toxin when bound to the AChR. The decrease in transfer efficiency upon cholate solubilization is not a consequence of either an altered overlap integral or donor quantum yield. Furthermore, based on the similarity of electron micrographs of the AChR in the mem- brane-associated and solubilized states (4-6, 11-16,35,36), it is unlikely that solubilization could significantly perturb the structure of the AChR to produce the lo-16-A intersite dis- tance change dictated by the difference in specific energy transfer between membrane-associated and solubilized states. Additionally the donor-occupancy dependence of the observed energy transfer efficiency does not fit expected energy transfer between the two sites, while the donor-occupancy dependence for the solubilized receptor does fit the model. Finally, it is unlikely that the observed energy transfer in solubilized AChRs stems from energy transfer between monomers of a disulfide-linked dimer, because exposure of the AChR to dithiothreitol prior to solubilization did not affect energy transfer (data not shown).

We have developed an approach which takes advantage of the slow dissociation rate of the toxins, particularly when bound to the membrane-associated AChR, and of the equal probability with which toxins bind to each site. To do so, we have made several assumptions. First, energy transfer is as- sumed not to occur between two FITC-toxins bound to the same AChR. This is a reasonable assumption because the overlap integral between FITC-toxin emission and absorption spectra is less than one-tenth of the overlap integral between FITC-toxin emission and TRITC-toxin absorption spectra (2.9 X compared to 1.9 x cms/mol). This means, for example, that if two FITCs were 67 A apart, then the transfer efficiency would be 0.6% (with K 2 = %) compared to 14% between FITC and TRITC. Another assumption we made was that the acceptor-TRITC-toxin was not excited directly at the donor excitation wavelength. This, too, is reasonable because excitation of TRITC-toxin (250 nM) at 450 nm never contributed more than 0.05% to the peak FITC- toxin emission at 519 nm, a value well within instrumental noise. Also, light absorption by the TRITC-toxin was assumed to be minimal at the peak FITC-toxin emission (519 nm). The extinction coefficient of TRITC at 519 nm is less than 30,000 M" cm" so that at 250 nM of TRITC-toxin (a typical concentration utilized), the per cent reduction in transmission is <I% from the center of the cuvette. The apparent reduction in FITC-toxin fluorescence due to light absorption of TRITC- toxin in the cuvette is, therefore, also 4 % . Moreover, the error produced by TRITC-toxin absorbance is not present in the calculated transfer efficiency, since it is subtracted from total transfer to yield the specific transfer efficiency.

To assess the contribution of nonspecific energy transfer, we incubated native toxin with the FITC-toxin-AChR com- plexes for typically 50 min (30 min before and 20 min after the addition of TRITC-toxin) to prevent the specific binding of TRITC-toxin. FITC-toxin fluorescence was then measured. In our calculations of transfer efficiency, we assumed that in this 50-min period no FITC-toxin dissociated. However, based on the dissociation rate constant of FITC-toxin (251, approx- imately 9% of the FITC-toxin would dissociate and be re-

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Energy Transfer between a-Toxin-binding Sites 5723

placed by native toxin. The observed FITC-toxin fluorescence should, therefore, decrease about 4.4% from FITC-toxin dis- sociation alone. This, of course, yields a lower estimate of the nonspecific transfer efficiency.

Since dipolar energy transfer shows a reciprocal sixth power dependence on interfluorophore distance, our findings enable US to place certain constraints on intersite distances. The largest component of energy transfer is intermolecular but since it is likely that this component involves multiple donor- acceptor pairs, intersite distances would depend on an esti- mate of available a-toxin sites approaching the critical trans- fer distance. This should depend on both the packing density of receptors in the membrane and their orientations with respect to each other.

Intramolecular transfer should result from a single type of donor-acceptor pair and the abundance of the hybrid species containing dissimilar toxin molecules may be estimated from the equations derived in the Appendix. The small values for intramolecular energy transfer yield intersite distances in excess of 55 A. Studies on the position of angular orientation of biotonyl-a-toxin indicate that the a-toxin molecules are not diametrically opposed (14). Taking these findings into consideration, the chromophores appear to be located on the outer perimeter of the receptor which has an overall diameter of 90 A. The uncertain position of lysine 23 on the donor with respect to the entire a-toxin-binding site and the absence of site-specific labeling of the TRITC-toxin present distinct limitations on a more precise assignment of distance between the a-toxin molecules. However, should microscopic hetero- geneity exist for the TRITC-toxin conjugate, intersite dis- tances would be represented by (ZiR?). The effect of this heterogeneity is to give greater weight to the contribution of the more proximal donor-acceptor pairs giving rise to higher energy transfer efficiency than would be obtained from an arithmetic average of the distances. Hence, if heterogeneity of labeling exists the average distance between donor-acceptor pairs would be greater than that calculated from the data. Thus, the distance estimates for energy transfer with the receptor oligomer and the substantial transfer between indi- vidual receptor molecules within the membrane indicate that chromophores exist near the outer perimeter of the receptor rather than being confined to the vicinity of a central axis (i .e. the internal cavity) perpendicular to the plane of the membrane.

We have presented here a framework with which the steady state fluorescence energy transfer technique can be utilized to explore the spatial relationship between two a-toxins bound to the AChR. Several strategies should allow future refine- ments of the estimates of intersite distances. The first will be the development of site-specific labeled a-toxins which can serve as acceptors. The congener to fluorescein, erythrosin isothiocyanate, is an attractive candidate for this purpose. Although erythrosin has a low quantum yield and measure- ment of acceptor sensitization will consequently be difficult, its overlap integral with fluorescein exceeds that of the TRITC and thus should prove useful for estimating energy transfer over longer distances. With two different sites spe- cifically labeled through a process of triangulation, the three- dimensional picture of the juxtaposition of the two toxins bound to the AChR can be drawn. Additionally, several fluo- rescent agonists and antagonists have been developed in recent years for study of the receptor; some of these agents possess the requisite specificity for energy transfer measure- ments. For example, we recently examined the interaction of a fluorescent analog of suberyldicholine in which the fluores- cent nitrobenzo-2-oxa-1,3-diazole is located between the two

quaternary ammonium moieties (37). Fluorescence energy transfer measurements between the bound ligand and the fluorescent toxin should proffer a fixed and unambiguous position for the chromophore with respect to the ligand- binding sites.

Acknowledgments-We wish to acknowledge the technical assist- ance of Blake Perkins for preparation of AChR membrane fragments, and Sandy Dutky for typing the manuscript.

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D A Johnson, J G Voet and P Tayloracetylcholine receptor.

Fluorescence energy transfer between cobra alpha-toxin molecules bound to the

1984, 259:5717-5725.J. Biol. Chem. 

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