N-(3-pyrene)maleimide: A fluorescent probe of acetylcholine receptor · Triton X-100 aggregates

ARCHIVES OF BIOCHEMISTRY Vol. 190, No. 1, September,

AND BIOPHYSICS pp. 57-66, 1978

N-(3-Pyrene)maleimide: Receptor.

A Fluorescent Probe of Acetylcholine Triton X-l 00 Aggregates’

vEsNA

SATOR, MICHAEL A. RAFTERY,~ MARINO MARTINEZ-CARRION3* 4

Department Virginia

of Biochemistry, Medical College of Virginia, 23298, and Department of Chemistry, University Received

December

Virginia Commonwealth of Notre Dame, Notre

28, 1977; revised

April

AND

University, Richmond, Dame, Indiana 46556

4, 1978

Acetylcholine receptor (AcChR) contains a disulfide bond which upon reduction can be labeled with a cholinergic analog, 4-( N-maleimide)a-benzyltrimethylammonium (MBTA) (Karlin, A. and Cowburn, D., 1973, Proc. Nat. Acad. Sci. USA 70,3636-3660). A fluorescent reagent, N-(3-pyrene)maleimide, which reacts preferentially with protein thiols (Wu, C. W., Yarbrough, L. Z. and Hsiuch, Y., 1976, Biochemistry 15, 2863-2868), has been introduced into solubilized preparations of Torpedo califorkca AcChR. Both freshly isolated receptor and N-ethylmaleimide (NEM) (to protect exposed SH groups) and dithiothreitol (DTT) (to reduce the cholinergic site disultide) treated receptor can be labeled with N-(3pyrene)maleimide (PM). The attachment of the fluorescent probe to AcChR does not perturb the binding of a-bungarotoxin (a-Bgt) or of a cationic site ligand, propidium (Sator, V., Raftery, M. A., and Martinez-Cation, M., 1977, Arch. Biochem. Biophys. 184, 95); it does, however, prevents labeling with MBTA. After treatment of AcChR with NEM and DTT, a-Bgt affords some protection against PM labeling. The PM probe inserted after reduction of the disulfide appears to rest on a cationic region(s) of the protein. Scrutiny of the fine vibrational structure, the total fluorescence or lifetime of the singlet excited state of bound PM show that binding of cholinergic ligands to AcChR does not alter the probe’s environment. Fluorescence polarization parameters of bound PM gauge the extent of detergent receptor interaction in solubilized preparations. From the calculated correlation times a molar volume of 9.57 X lo” is obtained for AcChR Triton X-100 complexes which correspond to average Stokes’ radii of 72A. The latter values provide an explanation for the large discrepancy between the molecular weight of the receptor protein (270,000) and its anomalous behavior in gel filtration columns

It has been hypothesized choline receptors, involved

(1) that acetylin the transmis-

sion of nerve impulses at the neuromuscular junction, undergo a structural transition upon interacting with acetylcholine, which in turn induces ion fluxes across the muscular membrane. It also appears that AcChR4 contains a large number of sulfhydry1 groups (2) and that reduction exposes other SH group(s) near the transmitter binding site (3,4). The availability of these chemical handles facilitates the insertion of spectroscopic probes covalently attached to the reduced disulfides. Such probes could improve the monitoring of ligand binding to nearby sites or to those which, through long range effects, would perturb the environment of the strategic sulfhydryl groups. Recently, a compound N-(3-pyrene)maleimide, has been reported (5,6) to form fluorescent adducts with organic com-

’ This work was supported by a grant-in-aid from Miles Laboratories (M.M.C.) and NIH research grants NS-10294 (to M.A.R.) and GM-24885 (to M.M.C.). ’ Permanent address: Division of Chemistry, California Institute of Technology, Pasadena, CA 91125. ” NIH Career Development Awardee. To whom to address correspondence at the Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298. 4 AcChR, acetylcholine receptor; PM, N-(3-pyrene)maleimide; NEM, N-ethylmaleimide; DTT, dithiothreitol; a-Bgt, a-bungarotoxin; d-TC, d-tubocurarine; Carb, carbamylcholine; SDS, sodium dodecyl sulfate; Deca, decamethonium; DTNB, 5,5’dithiobis(2-nitrobenzoic acid); MBTA, 4-( N-maleimido)a-benzyltrirnethylammonium; DATD, N,N-diallyltartar diamide; PMSF, phenylmethyl-sulfonylfluoride. 57

0003-9861/78/1901-0057$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved

58

SATOR,

RAFTERY,

AND

pounds and proteins containing sulfhydryl groups. Analysis of the fine vibrational structure of fluorescence emission of pyrene probes should provide information regarding the nature of the environment of PM. Furthermore, the long lifetime (- 100 ns) of the first excited singlet state of this fluorophore is also more than adequate to measure rotational relaxation times (7) of possible AcChR . Triton X-100 complexes. In this paper, we report the covalent marking of the AcChR with PM into easily accessible protein groups and into those amino acid residues which are expected only after reduction with DTT. The accessibility of AcChR-bound PM to outside (solution) quenchers is studied by fluorescence dynamic techniques in the presence and absence of some cholinergic ligands. In addition, fluorescence polarization and independent lifetime measurements of the singlet excited state are employed to investigate some properties of the AcChR-detergent complex( es). EXPERIMENTAL

PROCEDURES

Materials. [‘4C]N-(3-Pyrene)maleimide (as well as nonradioactive compound) was synthesized according to the procedure of Weltman et al. (5). The radioactive component was [1,4-‘4C]maleic anhydride (Amersham/Searle Corporation) and the specific activity of the compound was 0.8 mCi/mmol. a-Bgt was purified from Bungurus multicinctus venom (obtained from Sigma) following the procedure of Clark et al. (8). All the chemicals used were commercial analytical reagents. Acetylcholine receptor activity and purification from Torpedo californica electric organ was carried out according to published procedures (9, 10). Determination of SH groups of AcChR. The amount of accessible SH groups in AcChR was determined with DTNB (11). The SH group content was measured with freshly isolated (native) protein and in protein after overnight incubation in 2% SDS. When the receptor was reduced with 0.2 mM DTT prior to reaction with DTNB, oxidation of SH groups was prevented by working under dry nitrogen. Affinity labeling with MBTA. Labeling of the receptor with MBTA was performed according to the procedure of Karlin et al. (12) and the extent of labeling determined using ]‘H]MBTA. Labeling of AcChR with PM. AcChR was labeled with PM by adsorbing this fluorophore (100 molar excess over AcChR concentration) from a CHCl., solution on the walls of a test tube, adding the appropriate amount of AcChR (in 10 mM Tris-Cl, pH 7.4 buffer, containing 0.03% Triton X-106 and 0.02%

MARTINEZ-CARRION NaN:,) and stirring for a desired period of time (30 min to 10 h) at room temperature. The reaction was stopped by addition of mercaptoethanol in 100 molar excess over PM concentration. The suspension was filtered through a 0.45 p Millipore filter to remove undissolved PM, and the samples passed through Sephadex G-75 column (0.9 X 50 cm) to remove the small molecules. When binding protection was studied, AcChR was preincubated with a-Bgt for 1 h at room temperature and the receptor subsequently reacted with PM. Protection of exposed SH groups with NEM was carried out by incubation of AcChR with 100 molar excess NEM for 4 h in the 10 mM Tris-Triton buffer, pH 7.4. AcChR is removed from this mixture by passage through a Sephadex G-75 (0.9 x 50 cm) column. When the disulfide groups of the AcChR were reduced prior to labeling with PM, the procedure given by Karlin and Cowburn (12) was used. The reducing agent was always separated from AcChR by passage of the reaction mixture through a Sephadex G-75 column (0.9 x 50 cm). The extent of AcChR labeling with PM was determined by measuring the amount of radioactivity incorporated into protein (Packard, TriCarb liquid scintillation spectrometer) and by determining the absorbance of covalently attached PM at 343 nm (e = 4 x IO4 iv-‘cm-‘) (6). Since the two methods gave identical results the extent of labeling was routinely determined by the absorption at 343 nm. Absorption spectra and circular dichroism measurements of PM attached to the receptor were measured in a Cary Model 15 spectrophotometer and a Cary Model 60 spectropolarimeter after separation of the labeled AcChR from free PM by passage through a Sephadex G-75 column. The emission spectra as well as fluorescence titrations were carried out in a PerkinElmer MPF-44 or Aminco-Bowman spectrometers using l-cm cells and a thermostated cell holder. Lifetimes of fluorescence of pyrene maleimideAcChR deriuatiue. The lifetimes of fluorescence of pyrene malemide covalently bound to the receptor, were measured in deaerated samples using the fast kinetic spectroscopic methods described by Gratzel and Thomas (13) and Cheng et al. (14). The excitation wavelength of frequency doubled Q-switched laser was 374.1 nm and the fluorescence emission was followed at 400 nm. Polyacrylamide SDSgel electrophoresis of ‘%-PM labeled receptor. The distribution of the radioactive label on the subunits of AcChR was determined by SDS polyacrylamide disc gel (7%) electrophoresis (15) with the variation in crosslinking agent; instead of bisacrylamide, DATD was used (16). This allowed the dissolution of the gel in 2% periodic acid (0.4 ml per 0.2-0.5 mm gel slices), and efficient counting of radioactivity. The position of the protein bands on the gels was determined, after staining with Coomassie blue in a Photovolt recording densitometer using a 570-nm filter.

PYRENE

MALEIMIDE

IN

ACETYLCHOLINE

Peptide analysis of PM labeled AcChR. Boiled PM labeled AcCbR was digested for 12 h at 25°C with TPCK-trypsin (3O:l AcChR/Trypsin ratio) in 0.1 M Nethylmorpholine acetate buffer, pH 8$. After concentration by rotary evaporation the peptides were subjected to high voltage electrophoresk in Whatman 3 MM paper at pH 4.6 in pyridine-acetic acid-water (25:25:2950) buffer. After drying the papers were counted for radioactivity arising from [14C]PM and stained for ninhydrin positive spots. Fluorescence polarization analysis. Measurements of fluorescence polarization of the AcChR were carried out after labeling with PM. Measurements were performed with freshly isolated AcChR (10 mM Tris-HCI, pH 7.4, 0.03% Triton X-100, 0.02% NaNJ or after denaturation with 0.1% SDS, at 55°C for 10 min, prior to removal of Triton X-100 by extensive dialysis against 0.1% SDS. The buffer used was 0.1 M sodium phosphate, pH 7.0,0.1% SDS, 0.02% N&. The degree of fluorescence polarization P (defined as (I, -ZL)/(Zl + ZJ where 111 and I, are the intensities parallel and perpendicular to the polarized excitations) measurements were performed at various temperatures utilizing an Elscint Model MV-la fluorescence polarization apparatus. The excitation wavelength was 365 nm, and for emission the cut-off filter for wavelengths below 418 nm was used. All the samples were millipored and diluted until no further change of polarization was observed, to avoid light scattering and concentration quenching effects. Viscosities of the solvents were determined by utilizing the Ostwald viscosimeter. Rotational relaxation times (Ph) were obtained from the Perrin’s relation for a spherical molecule l/P - l/3 = (~/PO - l/3) (1 + (RT/qV) T,,), where ph = 37V/RT. Here P is a measured polarization, PO is the limiting value of polarization when no depolarization by molecular motion takes place, R is the gas constant, 2’ the absolute temperature, TJ the viscosity of the solvent, V is a parameter relating to the molecular shape of the fluorescent probe, 70 is the lifetime of the excited state of the fluorophore coupled to the protein molecule (measured as described above). The rotational relaxation time of the macromolecule is obtained from the slope of the plot of (l/P - l/3) vs. T/q.

RESULTS

SH group content by DTNB titration. In freshly isolated receptor about 5 SH groups/mole readily react with DTNB while about twice as many react after denaturation with 2% SDS (Table I). The total number is subject to sma.lI variations with different preparations which is similar to findings with AcChR from electrophorus (17) and with AcChR from two Torpedo species (2). Reduction of the receptor with 0.2 mM DTT when, presumably, an S-S bridge in the vicinity of the acetylcholine binding site is reduced (3)) should expose additional SW groups. The receptor is first covalently labeled by treating it with 100 molar excess NEM for 4 h at room temperature and removing unreacted NEM by passage through a Sephadex G-75 column. After DTT reduction of NEM treated receptor and removal of DTT from the medium by passage through another Sephadex G-75 column, there is additional exposure of SH residues. Under these conditions, 2 SH groups react with DTNB (Table I). Labeling of AcChR with N-(3-pyrene)maleimide. The fluorescent reagent N-(3-pyrene)maleimide conjugates with SH groups of proteins and smalI molecules like cysteine (5). Under nonreducing conditions between 5 and 9 sulfhydryls on AcChR should be accessible to PM and after reduction of AcChR with DTT two additional residues should become accessible (Table I). Treatment with NEM should protect all of the SH groups capable of reacting with DTNB or PM. After reaction of native AcChR with PM a slightly higher number of PM mol-

TABLE NUMBER

OF REACTIVE

Receptor

SH GROUPS

PER MOLE

No. of SH before Native

Native NEM-treatedb

5.5 + 0.3 0.3 f 0.25

59

RECEPTOR

OF AcChR

I AS DETERMINED

reduction In 2% SDS 8.7 + 0.2 0.8 + 0.25

n Reduction is performed with 0.2 mM DTT, for 10 min. b AcChR treated with 100 molar exces of NEM for 4 h; unreacted Sephadex G-75 column.

BY REACTION No. of SH after Native

7.7 k 0.25 2.3 + 0.3 NEM

removed

WITH DTNB reduction’ In 2% SDS 10.8 + 0.2 2.2 f 0.3

by passage

through

a

60

SATOR, RAFTERY,

AND MARTINEZ-CARRION

ecules/mole of AcChR are incorporated than had been expected (Fig. 1 and Tables II and III). The amount of PM bound to the receptor is time dependent and saturation is achieved within 7 hours (Fig. 1) when it reaches a maximum of 13.5 mol PM bound per receptor molecule. This number, however, varies slightly from preparation to preparation. I

I

I

HOURS

1. Binding of PM to native AcChR, followed as the amount of radioactivity incorporated into the protein. Specific activity of the receptor is 9 run01 of a-Bgt hound/mg protein.

Receptor

TABLE II ‘251-~-Bgt TO PYRENE MALEIMIDE ACETYLCHOLINE ‘9-a-Bgt bound

RECEPTOR Percentage of a-Bgt

III

AcChR

Native’ NEM-treated

Pr;Etfx

None cY-Bgt None a-Bgt

Mole;c%yRmole 2 h”

4 h”

10.0 9.1 3.0

13.5 13.0 6.4 3.7

‘Amount of PM incorporated measured after carrying out the incubation the number of hours specified and subsequent treatment of the samples with SDS and acetone as described by Weil et al. (4) for the labeling of AcChR with MBTA. ’ Freshly isolated and treated with PM. ‘Receptor treated first with NEM, reduced with DTT, and labeled with PM.

1

FIG.

BINDING OF LABELED

TABLE

LABELING OF AcChR WITH PYRENE MALEIMIDE: PROTECTION WITH WBUNGAROTOXIN

No. of

PM/receptor 3.8 5.3 6.5 9.8

~CDd Native 4680 100 NEM, PM 2 h” 4185 89.4 NEM, PM 4 h* 4371 93.4 93.2 PMLh 4350 92.8 PM4h 4345 n.* Receptor treated with 100 molar excess of NEM for 4 h (unreacted NEM removed on a Sephadex G-75 column) reduced with 0.2 roru dithiothreitol according to the procedure of Weil et al. (4) and treated with PM for 2 (a) and 4 h ( b) prior to the determination of ‘251-a-Bgt binding. ’ The receptor labeled with PM (no prior treatment with NEM and DTT), and binding of lz51-(u-Bgt to the PM-receptor complex tested.

Pretreatment of AcChR with an excess of NEM produces limited protection against further incorporation of PM (Tables II and III). In freshly isolated AcChR, a-Bgt affords an even smaller amount of protection than NEM to PM labeling. On the other hand, if the accessible SH groups of the receptor are blocked with NEM, preincubation with a-Bgt affords a 50% protection to PM labeling (Table III). Interestingly, [3H]MBTA which reacts with PM labeled AcChR after DTT treatment (data now shown) fails to do so if the exposure to MBTA is carried out in preparations where PM was added after DTT reduction of AcChR. Such behavior indicates a preference of MBTA and PM for the groups in the AcChR which become accessible to alkylating agents only after DTT reduction. PM, when bound to AcChR, shows absorption bands at 313,327, and 343 nm and fluorescence emission maxima at 374, 385, and 394 nm (Fig. 2). The absorption bands are void of dichroic effects. In other proteins the PM-protein fluorescent adducts undergo spectral changes in both absorption and emission within several hours after labeling, the phenomenon being explained as the maleimide ring opening as a result of hydrolysis of the probe (6). Using [14C]PM, the amount of radioactivity and/or fluorescence incorporated as number of moles of PM bound/mg AcChR is the same for both freshly labeled

PYRENE

MALEIMIDE

02~20 ‘loo 300

350

IN

ACETYLCHOLINE

400

nm

FIG. 2. Absorption (A) and emission (B) (excitation at 343 nm) spectra of PM covalently bound to the acetylchohne receptor (8 mol PM/mol AcChR). Buffer is 10 mM Tris/Triton X-100, pH 7.4.

preparation of AcChR or after their treatment with SDS. The latter procedure was carried out by dialysis against 0.1% SDS in 10 mu sodium phosphate, pH 7.4 (to remove possible unreacted PM or Py-NH, produced by hydrolysis of this compound) and passage through a Sephadex G-25 column (1.5 X 28 cm) equilibrated with the same buffer. Furthermore, AcChR labeled with PM does not show time dependent transformations of the spectra. Neither absorption nor fluorescence emission spectra change either in shape or intensity over a period of l-2 weeks. Thus, little or no [‘%]PM appears to be hydrolyzed into aminopyrene and a radioactive succinyl moiety bound to the SH group on the receptor. Possible perturbation of the receptor’s capability of binding ligands after extensive labeling with PM was tested by the ability to bind propidium, which is a compound specific for one of the AcChR cationic binding sites (18). The receptor was first reacted with NEM for 4 h to protect against labeling with PM, reduced with DTT (4,12) and reacted with PM for another 4 h (6.6 mol PM bound/m01 AcChR). Titrations of this preparation with propidium, following its fluorescence at 623 nm (excitation at 545 nm), showed no change of the ability of the PM labeled receptor to bind this compound. The Kd value for propidium with

RECEPTOR

61

the native protein was 1.3 x lo-“M, and after PM labeling 1.1 X 10e6 M. Also the ability of the receptor to bind [‘251]a-Bgt, after reacting with PM with or without pretreatment with NEM (100 molar excess, 4 h) and subsequent DTT reduction, is virtually unaltered (Table II). These results agree with our previous findings that labeling of the strategic sulphydryl with the affinity label MBTA did not perturb the affinity of AcChR for propidium or with the report that in Electrophorus preparations treatment of AcChR with a variety of SH blocking reagents did not perturb the receptor’s affinity for a-Bgt (17). Location of Pit4 binding. Distribution of the radioactively labeled PM on the subunits of AcChR was determined by polyacrylamide gel electrophoresis in SDS. Figure 3 shows the distribution of the radioactivity on the gels. The peak with highest radioactivity content coincides with the 40,000-dalton subunit. However, a small and reproducible amount of radioactivity is also found in the 52,000- and 58,000-dalton subunits. With prolonged time of labeling, both of the latter peak heights increase, yet the amount of radioactivity incorporated into the 40,000 subunit is always the largest. Radioactivity arising from PM labeled AcChR peptides was distributed among several ninhydrin positive spots of the paper electrophoresis pattern of tryptic digests of preparations labeled with PM, with or without pretreatment with NEM (data not shown). Fluorescence quenching constants. Fluorescence of receptor-bound PM is characterized by only one lifetime of 106 f 5 ns (excitation wavelength was 347.1 nm, emission 400 nm).” Information regarding the microenvironment of incorporated PM, as ’ Three to four weeks after labeling a component with short fluorescence lifetime (38 + 5 ns) appears and is attributed to a possible hydrolysis of the receptor since it was also observed that, in SDS polyacrylamide gel electrophoresis, the radioactivity incorporated into 40,000 subunit as well as the protein stain intensity decreased upon standing and a new, faster moving band of protein containing radioactivity appears. The appearance of the band and of the shorter lived fluorophore can be largely prevented by treatment of the AcChR preparations with PMSF.

62

SATOR, I

I

RAFTERY,

I

I

AND

I

15cIE 8 IOC)\

MARTINEZ-CARRION

the receptor are compared in Fig. 4 and Table IV. TlN03 and CHsNO2 are equally efficient and better quenchers of fluorescence of PM bound to the receptor than II. The rates of fluorescence quenching for PM in solution are significantly higher than those for PM in the receptor. Furthermore, free PM solutions do not significantly discriminate among the various quenchers tested.

5CII

I

I

I

I

2

4

6

0

IO

cm FIG. 3. Distribution of radioactive PM on the subunits of AcChR determined by polyacrylamide disc gel electrophoresis in 0.1% SDS. Top diagram shows the Coomassie blue stain; Bottom, the radioactivity tracing.

well as of its exposure to solvent medium, can be gathered, by analysis of the fluorescence quenching effects of external additives: positive or negative ions, or neutral molecules (14). Decay of fluorescence of the excited state is facilitated by these added solutes and the quenching of the excited singlet state by the external molecules follows the Stern-Vohner equation. +o/$

=

1 +

k2

TO[&]

=

TO/T

Where + and +O are fluorescence quantum yields in the presence and absence of quencher, 7. fluorescence lifetime in absence of quencher, [Q] is the concentration of the quencher, and k2 is the bimolecular quenching constant. The constant of fluorescence quenching by external additives gives information on the easeof approach of that particular molecule to the fluorophore. The nature of the quencher can also provide information on the microenvironment, and the exposure of the probe covalently bound to the receptor. The quenching data can be obtained by measuring either fluorescence intensities or fluorescence lifetimes 7 (since 9 = T/T~) at different quencher concentrations. In this study the quenchers used are T1N03, KI, and CHzN02. The ease of quenching of PM in solution (10 mM Tris-Cl, pH 7.4, 0.03% TX-100, 0.02% NaN3) and when bound to

2 FO F

I

I

I

20

1

40 (KIl

60

X IO:

M

FIG. 4. Stern-Volmer plots of the KI-induced quenching of fluorescence of PM covalently attached to the AcChR (M), and of PM alone (U---U) both in 10 loll Tris-Cl buffer, pH 7.4 containing 0.03% Triton X-100 and 0.02% NaN3. Receptor is labeled with 3 PM mol/mol AcChR after pretreatment with NEM and reduction with DTT. PM concentration is 3.5 CM. TABLE

IV

QUENCHING CONSTAN-&’ OF FLUORESCENCE PYRENE MALEIMIDE ON THE ACETYLCHOLINE RECEPTOR AND IN SOLUTION Quencher

k,16 (M-I s-') PM-receptor

TlNOa CHBNOZ KI

k,Z' (M-' 8) PM-buffer

x10m8

x10-R

3.99

9.34

2.43 0.76

6.33 3.98

OF

k,Z/k,l 2.3 2.6 5.2

u Lifetime of PM bound to the receptor is 110 ns, and of PM in buffer 79 ns. * Quenching constant for the PM covalently bound to the receptor in the ratio 3:l. ‘Quenching constant for the PM dissolved in Tris/Triton X-100 buffer, pH 7.4.

PYRENE

MALEIMIDE

IN

ACETYLCHOLINE

The possible perturbation of the bound PM environment by the presence of the choline& ligands was also examined. Figure 5 shows the rate of fluorescence decay where Tl’ is used as a quencher in the presence and absence of 10e4 M carbamylcholine (concentration 10 times higher than its &). The rate of the process was followed by directly measuring the lifetimes of PM fluorescence as indicated in the Methods section. The two quenching constants (3.7 x 10’ M-’ s-j in the absence and 3.1 x 10” M-’ SK’in presence of Carb) are of the same magnitude and are similar for all ligands tested (Deca, a-Bgt, d-Tc, Carb). The results obtained from the fluorescence dynamic studies were the same in AcChR preparations labeled with PM immediately after isolation or after reaction of the receptor with excess NEM and reduction with DTT. In all cases, there is no detectable perturbation at the region of the receptor where the PM is situated. Presence of increasing concentration of AcChR ligands, agonists and antagonists of acetylcholine (Carb, Deca, d-Tc, cu-Bgt), do not perturb either the intensity or vibrational frequency of the fluorescence maxima of PM bound to AcChR where the AcChR is randomly labeled with PM or after the more selective labeling of the SH groups assumed to be in the vicinity of the acetylcholine binding site. Identical results are obtained when the label is limited (3 PM/AcChR) or when it is fairly extensive (10 PM/AcChR). Lifetimes of AcChR bound PM fluorescence (in the absence of external quenchers) did not change with increasing concentrations of the above mentioned cholinergic ligands. Fluorescence polarization studies of PM-AcChR complex. Knopp and Weber (19) have suggested that for the fluorescence polarization studies of large molecular structures (Mr - 10”) fluorescent probes of relatively long fluorescence lifetimes (100 ns) should be used, since the lifetime of the conjugate determines the range of rotational relaxation times that can be measured. PM, which has a long fluorescence lifetime when conjugated with a protein, should be a probe suitable for fluorescence polarization studies of a large macromol-

63

RECEPTOR

T 1.2 ” PC 4 x * 0.8’

I

I

I

2

4

6

(TIN03)

I

X 103,M

FIG. 5. TIN03 induced quenching of fluorescence of PM, covalently bound to AcChR in absence (G--Q and presence (l&--U) of 10m4 M carbamylcholine. Same receptor preparations as in Fig. 4.

ecule such as AcChR. From hydrodynamic studies of the AcChR molecule (20, 21) it was suggested that this protein has the same Stokes radius (73 A) as P-galactosidase (Mr = 520,000), whereas its sedimentation coefficient is abnormally small (9 S) compared to that for P-galactosidase (15 S). On the other hand, the molecular weight of AcChR appears to be about 300,000 (22, 23). The receptor also shows a tendency to bind large amounts of Triton X-100 when solubilized in this detergent (about 45% of its total weight) (22). Hence, it is likely that the hydrodynamic anomalities of the receptor may be derived from its tendency to interact with large amounts of Triton X100 which contribute to the size and/or symmetry of the structure. Figure 6 shows the results of fluorescence depolarization measurements of PM-receptor conjugates by exciting the complex at 365 nm and varying the temperature of the sample in the range of 5-35’C.” The fluorescence polarization measurements were performed with preparations of AcChR in which the fluorophore/receptor ratio was 5.6 or 12, with no indication of the dependence of polarization on the value of this ’ Sucrose or glycerol could not be used to vary the viscosity of the solution because high concentrations of sucrose caused precipitation of Triton X-100 and receptor protein, while solutions containing increasing concentrations of glycerol gave a negative slope of the (l/P-1/3) vs. T/q plot.

64

SATOR,

RAFTERY,

AND

ratio, PO being the same (0.277) for both. Within the range of temperature studied, the results do not indicate complexity in the rotational freedom of the conjugates. From our independently determined value of 111 ns for the fluorescence lifetime, a rotational relaxation time of 1170 + 150 ns was calculated (Table V). The latter value is comparable with the rotational relaxation time of 1140 ns obtained for human macroglobulin (IgM) with the molecular weight of 890,000 (19). Assuming a spheric structure of the fluorophore-receptor complex in the Triton X-100 solution, the above value corresponds to particles with molar volumes with Stokes radii of 72 A, an identical value to that obtained by chromatography on Sepharose-6B columns (20). When the receptor is denatured with SDS, and Triton X-100 removed by extensive dialysis, there

1

30 kc-100

200 300 T/n X lO-2

400 (‘K/p1

500

FIG. 6. Plot of fluorescence depolarization studies of PM-AcChR conjugates: (0) AcChR in buffer (10 nuu Tris-Cl, pH 7.4,0.03% Triton X-100). (0) AcChR under denaturing conditions (0.1% SDS, 0.1 M phosphate buffer, pH 7.0). Chromophore/AcChR ratio is 5.6, 0.25 mg protein/ml. Excitation at 365 nm.

MARTINEZ-CARRION

is an appreciable decrease in the degree of fluorescence polarization. On the other hand, in SDS treated AcChR, the bound PM probe shows a fluorescence lifetime for the singlet excited state of only 68 ns and the rotational relaxation time is markedly decreased to a value of 333 f 35 ns. DISCUSSION

There is some discrepancy regarding the number of SH groups accessible to DTNB and PM reagents. About 5-9 SH groups are available for the reaction with DTNB and virtually all of these sulfhydryls can be protected with NEM (Table I). Nevertheless, the receptor binds more PM molecules than would correspond to the number of available SH groups (Fig. 1 and Tables II and III) and NEM does not fully protect against reaction with PM. This behavior could be explained if not all SH groups were blocked with NEM or DTNB, or more likely, if other groups (possibly NHz) on the AcChR became labeled with PM. However, regardless of the nature of the PM target sites, it is of interest that most PM seems to bind to the 40,000-dalton subunit. This subunit is known to bear part of the ligand binding sites for acetylcholine analogs (4, 12) and it must constitute an exposed part of the AcChR molecule. The pyrene succinimide moiety of bound PM could be situated on the surface of the receptor molecule in two different ways with the succinimide group exposed and pyrene ring buried or vice versa. Since the protein-probe conjugates show no circular dichroicity in the region of their absorption maxima, the pyrene moiety is not likely to be held rigidly in the chiral environment of the AcChR. The bulk of PM labeling under nonreducing conditions does not seem to occur in

TABLE SUMMARY

OF FLUORESCENCE

POLARIZATION

TO(ns) Native SDS

AcChR denatured

’ Receptor

AcChR has 5.6 PM

bound

STUDIES Molar volume (ml/mole)

V OF RECEPTOR-PYRENE

ph (ns)

1170 (+150 ns) 333 (f35 ns)

111

9.57 f lo5

72

68

2.56 + lo5

47

per molecule

of protein.

MALEIMIDE

Stokes radius (A)

CONJUGATES”

PO 0.277 0.320

PYRENE

MALEIMIDE

IN

ACETYLCHOLINE

the vicinity of the receptor binding regions since there is little a-Bgt protection to PM incorporation. On the other hand, when the receptor was first protected with NEM to block most of the random labeling (exposed SH residues) followed by DTT reduction of disulfides, a-Bgt protected about half the SH groups against PM labeling. It looks as though 3 PM molecules bind to regions of the AcChR which combine with a-Bgt and that in DTT reduced AcChR the groups binding MBTA and PM are identical. It is surprising, however, that a-Bgt does not affect the fluorescence properties of bound PM. It is conceivable that the fluorophore could lay in a pocket or cleft in the AcChR molecule; in such setting it could remain unperturbed by the binding of the a-Bgt peptide anchored to the edges or opening of such pocket or cleft. The toxin, nevertheless, could sterically hinder access of externally added PM or MBTA. This interpretation is consistent with findings in which isolated receptor labeled with MBTA retains most of its ability to bind toxin, yet, toxin prevents labeling of AcChR with MBTA (24). The quenching behavior of the PM probe provides some insight regarding the nature of the environment of PM on the receptor. Quenching studies indicate (Fig. 4 and Table IV) that the relative rates of accessibility of Tl’ and CH3N02 to PM are not affected by the AcChR molecule since the ratio of the quenching rates for free and receptor-bound PM are of the same magnitude. In contrast, the significant variation in the value of the ratio for I- quenching may indicate that PM rests on negatively charged regions of the receptor (which is a highly electronegative molecule) barraging PM from I- collisions. High concentrations of carbamylcholine do not influence the exposure of the fluorophore to quenchers. Moreover, fluorescence parameters, indicative of the environment of the probe (25), i.e., the fine vibrational structure and singlet fluorescence lifetimes, also remain unaltered in the presence of cholinergic ligands. Because there are no perturbations of the fluorescent parameters for either “randomly” or selectively labeled (at the reduced S-S site) AcChR preparations, it is

RECEPTOR

65

unlikely that the perturbations occur only in a small population of the PM molecules and, thus, remain undetected. (In selectively labeled AcChR, at least 50% of the probe molecules should have been perturbed.) A more likely explanation for lack of detection of carbamylcholine in the putative ligand induced comformation change is that detergent-solubilized Torpedo AcChR seems to be “locked” into the state that shows relatively low affinity for acetylcholine agonists (26,28); and at least one conformational state must be absent in our preparations of solubilized receptor. Thus, the absence of perturbations of the PMreceptor conjugates induced by cholinergic ligands, may simply indicate that no transition to a different conformational state occurs upon binding the ligands to that state peculiar to our solubilization procedure. Another possibility is that PM is bound to regions of the AcChR that are not effected by the ligand-receptor interaction and if the conformational transitions are minor or localized they could remain undetected. The PM probe provides new information regarding the size of the large structure of receptor-detergent complex. Fluorescence polarization studies reveal rotational relaxation times for the PM receptor conjugates similar to that of IgM (M.W. 890,000). Instead, the molecular weight of the receptor is closer to the molecular weight of the subunits of IgM (180,000). This is consistent with the notion of the receptor aggregates to a large extent with Triton X-100 (22). The great amount of bound detergent is an integral part of the large soluble structure and/or assymetry of the complex. This model is strengthened by the observation that SDS (which dissociates the receptor into its subunits) decreases both the rotational relaxation time and the calculated Stokes radius. Furthermore, since there is only one detectable rotational relaxation time in SDS solutions, and the 40,000 subunits are predominantly labeled with PM, the measured rotational relaxation time in SDS most likely corresponds to that of this AcChR subunit in the assymetric elongated form common for proteins in SDS (29). While this work was being written, a

66

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RAFTERY,

AND

report appeared regarding the presence of high (13 S) and low (9 S) sedimentation AcChR forms in Torpedo californica preparations solubilized with Renex-30 detergent (30). The distribution between high and low species was dependent on the amount of calcium present (30). We have not observed much heterogeneity in our sucrose density gradient patterns of Triton X-100 solubilized preparations of AcChR. The preparations used in this work consisted of molecular forms that exist in solution as a population of proteins in which 90% of the molecules sediment as 9 S particles. These 9 S particles have an average number molecular mass of 270,000 + 30,000 daltons (22) and were shown to contain a high amount of bound detergent (22). These same particles we now report to have an average radius of 72 A. REFERENCES 1. NACHMANSOHN, D. (1975) Chemical and Molecular Basis of Nerve Activity, Ed. 2, Vol. 1, Academic Press, New York. 2. ELDEFRAWI, M. E., ELDEFRAWI, A. T., AND WILSON, D. B. (1975), Biochemistry 14, 4304-4310. 3. KARLIN, A. (1973), Federation Proc. 32, 1847-1853. 4. WEIL, CH. L., MCNAMEE, M. G., AND KARLIN, A. (1974) Biochem. Biophys. Res. Commun. 61, 997-1003. 5. WELTMAN, J. K., SZARO, R. P., FRANCKELTON, JR., A. R., DOWBEN, R. M., BUTWING, J. R., AND CATHOWN, R. E. (1973) J. Biol. Chem. 248, 3173-3177. 6. WV, CH. W., YARBROUGH, L. Z., AND YING HSIUCH, F. (1976) Biochemistry 15, 2863-2868. 7. YGUERRABIDE, J. (1972) in Methods in Enzymology (Him, C. H. W., and Timasheff, S. N., eds.), Vol. 26C, p. 498, Academic Press, New York. 8. CLARK, D. G., MACMURCHIE, D. D., ELLIOTT, E., WOLCOTT, R. G., LANDEL, A. M., AND RAFTERY, M. A. (1972) Biochemistry 11, 1163-1668. 9. SCHMIDT, J., AND RAFTERY, M. A. (1973) Biochemistry, 12,852-856. 10. SCHMIDT, J., AND RAFTERY, M. A. (1973) Anal. Biochem. 52,349-354.

MARTINEZ-CARRION 11. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70. 12. KARLIN, A., AND COWBURN, D. (1973) Proc. Nut. Acad. Sci. USA 70,3636-3660. 13. GRATZEL, M., AND THOMAS, J. K. (1973) J. Amer. Chem. Sot. 95,6885. 14. CHENG, S., THOMAS, J. K., AND KULPA, C. F. (1974) Biochemistry 13, 1135. 15. OSBORN, M., AND WEBER, U. (1969) J. Biol. Chem. 266.4406-4412. 16. ANKER, H. S. (1970) FEBS Lett. 7, 293. 17. CHANG, H. W. (1974) Proc. Nat. Acad. Sci. USA 71,2113-2117. 18. SATOR, V., RAFTERY, M. A., AND MARTINEZ-CARRION, M. (1977) Arch. Biochem. Biophys. 184, 95-102. 19. KNOPP, J. A., AND WEBER, G. (1969) J. Biol. Chem. 246,6309-6315. 20. RAFTERY, M. A., SCHMIDT, J., CLARK, D. G., AND WOLCOTT, R. G. (1971) Biochem. Biophys. Res. Commun. 65, 1622-1629. 21. MEUNIER, J. C., OLSEN, R. W., MENEZ, A., FROMAGEOT, P., BOQREUT, P., AND CHANGEUX, J. P. (1972) Biochemistry 11, 12OO-1210. 22. MARTINEZ-CARRION, M., SATOR, V., AND RAF’TERY, M. A. (1975) Biochem. Biophys. Res. Commun. 65, 129-137. 23. EDELSTEIN, S. J., BEYER, W. B., ELDERFRAWI, A. T., AND ELDEFRAWI, M. E. (1975) J. Biol. Chem. 250,6101-6106. 24. KARLIN, A., WEILL, C. L., MCNAMEE, M. G., AND VALDERRAMA, R. (1976) Cold Spring Harbor Symp. Quant. Biol. 40, 203-210. 25. KALYANASYNDARAM, K. (1976) Ph.D. dissertation, University of Notre Dame. 26. RAFTERY, M. A., VANDLEN, R. L., REED, K. L., AND LEE, T. (1976) Cold Spring Harbor Symp. Quant. Biol. 40,192-202. 27. CHANGEUX, J. P., BENEDETTI, L., BOURGEOIS, J. P., BRISSON, A., CARTAUD, J., DEVAUX, P., GRUNHAGEN, H., MOREAU, M., POPOT, J. L., SOBEL, A., AND WEBER, M. (1976), Cold Spring Harbor Symp. Quant. Biol. 40.211-230. 28. LEE, T., WITZEMANN, V., SCHIMERLIK, M., AND RAFTERY, M. A. (1977) Arch. Biochem. Biophys. 183,57-64. 29. REYNOLDS, J. A., AND TANFORD, CH. (1970) J. Biol. Chem. 245,5161-5165. 30. CHANG, H. W., AND BOCK, E. (1977) Biochemistry 16,4513-4520.