Time-resolved Fluorescence Study of the Neuron-specific ...

THE JOURNAL OF BIOLOGKXL CHEMISTRY 0 1990 by The American Society for Biochemistry

Vol. 265, No. 21, Issue of July 25, pp. 12584-12595.1990 and Molecular

Printed in U.S.A.

Biology, Inc.

Time-resolved Fluorescence Study of the Neuron-specific Phosphoprotein Synapsin I EVIDENCE FOR PHOSPHORYLATION-DEPENDENT

CONFORMATIONAL

CHANGES* (Received

Fabio

Benfenati$QI[

11,Paolo

From the *Institute of Human University of Parma, Parma, New York, New York 10021

Neyrozll

**, Martin

Btihlerl,

Lanfranco

Masotti**,

Physiology, University of Modena, Modena, Italy, the **Znstitute Italy, and the §LaboratorN of Molecular and Cellular Neuroscience,

Synapsin I is a major nerve terminal-specific phosphoprotein. It consists of a hydrophobic head region containing one phosphorylation site for either CAMPdependent protein kinase or Ca2’/calmodulin-dependent protein kinase I and of a basic and elongated tail region containing two phosphorylation sites for Ca’+/ calmodulin-dependent protein kinase II. The steadystate emission spectrum of synapsin I was centered at 330 nm and was markedly red shifted upon denaturation, as expected for tryptophan residues segregated from the external aqueous environment in native conditions. Quenching studies showed a low accessibility of synapsin I tryptophans at low ionic strength which was further decreased by exposure to 200 mM NaCl but not significantly affected by phosphorylation. The intrinsic fluorescence of synapsin I was resolved into three major decay components with lifetimes of about 0.2, 3, and 7 ns. Upon phosphorylation of synapsin I on the tail sites, the spectra associated with the intermediate and long lifetimes were shifted to the red region, while the spectrum associated with the short lifetime was shifted to the blue region, in the absence of significant changes of the lifetimes. Phosphorylation of synapsin I on the head site was less effective. The anisotropy decay of synapsin I labeled with the longliving chromophore pyrene on Cys-223 was also analyzed. A shorter rotational correlation time was found for the tail phosphorylated form (corresponding to a Stokes radius of 41-42 A) than for the dephosphorylated or for the head phosphorylatgd form (corresponding to a Stokes radius of 60-63 A). The data suggest that phosphorylation of the tail sites induces changes in the conformation and hydrodynamic properties of synapsin I which may play a role in the regulation of the molecular interactions of synapsin I within the nerve terminal.

* This work was supported by NATO Collaborative Grant 0039/89 (to F. B., P. N. and-P. G.), United States Public Health Service Grants AA06944 and MH39327 (to P. G.), Minister0 Pubblica Istruzione 60% Grant (to F. B.), and Consiglio Nazionale delle Ricerche Progetto Finalizzato B.B.S. 89.00182.70 (to P. N. and L. M.). The co& of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. ll The first two authors contributed equally to the experimental work. j] To whom correspondence should be sent: Institute of Human Physiology, University of Modena, Via Campi 287, Modena I-41100, Italy. Tel.: 39-59374179; Fax: 39-59367372.

for publication,

and Paul

January

12, 1990)

Greengardg

of Biological Chemistry, The Rockefeller University,

Synapsin I, a collective name for two closely related proteins (synapsin Ia and Ib) with apparent molecular weights of 86,000 and 80,000 on SDS-polyacrylamide gel electrophoresis, is a major neuron-specific phosphoprotein that is concentrated in the presynaptic nerve terminal in association with the cytoplasmic surface of synaptic vesicles (l-5). Synapsin I has been purified to homogeneity from bovine and rat brain (6, 7), and its molecular structure has been characterized. It has a structure consisting of an NHz-terminal head region with a large proportion of hydrophobic (40%) and charged (27%) residues and of a COOH-terminal collagenasesensitive, elongated, and strongly basic tail region that contains a very high proportion of proline (27%), glycine (25%), and positively charged amino acids (10%) with only 2 negative residues (6, 8, 9). Synapsin I is highly surface active and is able to interact with phospholipid bilayers and to penetrate the hydrophobic core of the membrane (6, 10, 11). Synapsin I is an excellent substrate for several protein kinases in the brain and undergoes multisite phosphorylation. One serine residue in the head region (Ser-9, site 1) is phosphorylated by either CAMP-dependent protein kinase or Ca”/calmodulindependent protein kinase I, whereas 2 serine residues in the tail region (Ser-566, site 2 and Ser-603, site 3) are selectively phosphorylated by Ca’+/calmodulin-dependent protein kinase II (9, 12-14). The primary structures of synapsin Ia and Ib have been deduced recently from cDNA cloning (9). The two polypeptides, composed of 704 and 668 amino acids in the rat, are virtually identical with the exception of a small COOHterminal region. They arise from the same gene by differential splicing and are highly conserved evolutionarily. In uitro and in viuo experiments have demonstrated that synapsin I is involved in the modulation of neurotransmitter release through its reversible phosphorylation. Phosphorylation of synapsin I within the nerve terminal is stimulated by a variety of manipulations that increase neurotransmitter release such as electrical stimulation or depolarization (for review, see 15-17). Moreover, the microinjection of the dephosphorylated form but not of the phosphorylated form of synapsin I into the squid giant axon induces a marked and reversible inhibition of neurotransmitter release (18). In uitro studies have demonstrated that synapsin I binds with high affinity to synaptic vesicles and possesses F-actin binding and bundling activity. Both interactions are signifi’ The abbreviations used are: SDS, sodium dodecyl sulfate; NTCB, 2-nitro-5-thiocyanobenzoic acid; DTT, dithiothreitol; acrylodan, 6acryloyl-2-dimethylaminonaphthalene; NATA, N-acetyl-L-tryptophanamide; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; DAS, decay-associated spectra.

12584

Phosphorylation-induced

Conformational

cantly depressed by phosphorylation of synapsin I on the tail sites whereas phosphorylation of the head site is less effective (4, 7, 19). Structure-function analysis of synapsin I revealed that hydrophobic/amphiphilic domains of the head region (NH,terminal fragment) bind to acidic vesicle phospholipids and penetrate the membrane bilayer (lo), the tail region binds to synaptic vesicle protein(s) (ll), and a strongly hydrophobic fragment of the head region (middle fragment) interacts with F-actin (8) (see Fig. 1). The existence of distinct sites on synapsin I for synaptic vesicle and F-actin binding suggests that synapsin I may modulate neurotransmitter release by acting as a phosphorylation-dependent link between synaptic vesicles and the F-actin cytoskeleton of the nerve terminal (16, 17). In this work, we have attempted to investigate the effects of phosphorylation as well as of ionic strength on the occurrence of conformational transitions in the synapsin I molecule which may relate to its biological function. The intrinsic fluorescence of the 4 tryptophan residues present in the primary structure of synapsin I (Trp-126, Trp-227, Trp-335, Trp-356) as well as the fluorescence of synapsin I covalently labeled with the fluorescent sulfhydryl reagents N-(l-pyrenyl)maleimide and acrylodan have been characterized using steady-state and time-resolved fluorescence techniques. These spectroscopic techniques have been widely used as sensitive and valuable tools for monitoring conformational changes of protein molecules (20-24). The results obtained on the photophysical properties and the rotational relaxation kinetics of synapsin I provide new information on the microenvironment of tryptophan and cysteine residues and demonstrate the existence of conformational transitions in response to site-specific phosphorylation and ionic strength. EXPERIMENTAL

PROCEDURES

[T-~‘P]ATP (2,900 Ci/mmol) was obtained from Du Pont-New England Nuclear. Nonidet P-40, 2-mercaptoethanol, NTCB, DTT, NATA, and protease inhibitors were purchased from Sigma; guanidine hydrochloride was from Pierce Chemical Co.; Sephacryl S-200, from Pharmacia LKB Biotechnoloav Inc.: hvdroxvlaoatite, from BioRad; YM membranes for ultrafiltra‘tion, from AmGon (Dauvers, MA); Cm-cellulose (CM52), from Whatman; and Staphylococcus aureus V8 protease, from Miles Laboratories Inc. (Elkhart, IN). N-(l-Pyrenyl)maleimide and acrylodan were purchased from Molecular Probes Inc. (Eugene, OR) and used without further purification. Catalytic subunit of CAMP-dependent protein kinase was a gift of Drs. A. Horiuchi and A. Nairn of our laboratory. Calmodulin and Ca”/ calmodulin-dependent protein kinase II were gifts of Drs. F. Gorelick and G. Thiel of our laboratory. All other chemicals, obtained from Sigma or Serva Fine Biochemicals (Heidelberg, Federal Republic of Germany), were of analytical grade. Purification

and Phosphorylation

of Synapsin

I

Synapsin I was purified from bovine brain under nondenaturing conditions as described by Bahler and Greengard (19). Synapsin I was phosphorylated to near stoichiometry at (a) site 1 (head region) using the catalytic subunit of CAMP-dependent protein kinase; (b) sites 2 and 3 (tail region) using Ca*+/calmodulin-dependent protein kinase II; (c) sites 1, 2, and 3 using both kinases as described (7), except that detergent was omitted. After phosphorylation, synapsin I was repurified by batch adsorption to Cm-cellulose. A trace amount of [y-“‘P]ATP was added to the reaction mixtures for determining the stoichiometry of phosphorylation. The selective incorporation of 32P into head and tail regions was assessed by one-dimensional proteolytic phosphopeptide mapping using S. aureus V8 pro-

tease as described (13). Nonspecific than were

the specific 0.96, 2.03,

phosphorylation

at sites other

ones was less than 10%. The average stoichiometries and 2.72 mol of phosphate per mol of synapsin I for

12585

the a, b and c forms, respectively. A separate sample of synapsin I was subjected to the phosphorylation reaction without the addition of ATP

and used

in the experiments

N-(1 -Pyrenyl)maleimide

as dephosphosynapsin

and Acrylodan

Adducts

I.

of Synapsin

I

Prior to conjugation, purified synapsin I (0.75-1.0 mg/ml) was extensively dialyzed against a buffer containing 20 mM MOPS/ NaOH, pH 7.0, 100 mM NaCl, 1 mM EDTA in order to remove reducing agents completely. To prevent spontaneous oxidation of cysteine sulfhydryl groups, the dialysis buffer was degassed and saturated with Nz. For labeling synapsin I, 20 mM stock solutions of pyrenylmaleimide and of acrylodan were freshly prepared in dimethyl formamide. The reagents were then added to the protein solution to a final concentration of 200 pM (15-20-fold molar excess over syn-

apsin I), and the reaction was allowed to proceed for 8-12 h at 4 “C in the dark with constant rotation. After completion reaction, the samples were centrifuged in a minifuge ments Inc., Farmingdale, NY) at maximal speed for supernatant was batch adsorbed to Cm-cellulose for unreacted reagents. Labeled synapsin I was eluted

taining 200

mM

NaCl, 25

mM

Tris-HCl,

pH 8.0, 0.1

of the labeling (Savant Instru5 min, and the removal of the in a buffer conmM

EGTA and

stored in liquid Nz. The stoichiometry of labeling was estimated from the absorption spectra of the solution of protein adducts assuming a molar extinction coefficient of 4.05 X lo4 M-’ cm-’ at 345 nm for pyrene chromophore (25,26) and 1.29 X lo4 M-’ cm-’ at 360 nm for acrylodan chromophore

(27) in water using a Beckman DU-8 spectrophotometer.

Because of

the overlap between the absorption spectra of pyrene, tryptophan, and tyrosine (25), the concentration of synapsin I was determined by the method of Lowry et al. (28) using synapsin I as a standard. The concentration of synapsin I in the standard solution was assessed by means of the experimentally determined extinction coefficient at 277 nm (6). Pyrene-labeled synapsin I was subjected to site-specific phosphorylation by purified protein kinases as described above. No significant differences in phosphate incorporation were observed between unlabeled and pyrene-labeled synapsin I. To determine the location of the fluorescent label, repurified pyrene- and acrylodan-labeled synapsin I as well as mock-derivatized synapsin I were cleaved at free cysteine sites with NTCB following the procedures described previously (8, 29) with slight modifications. Samples containing 40 scg of synapsin I were prepared for hydrolysis by dialysis against-6 M guanidink HCl, 0.2 G Tris-HCl, pH 8.0, 0.1 mM

Materials

Changes of Synapsin I

EDTA, 2

mM

DTT and then against 6

M

auanidine HCl. 0.2

M

Tris-HCl, pH 8.0,O.l mM EDTA, o.fmM DTT.After dialysis, NTCB was added (5 mM final concentration) and the samples incubated at 37 “C in the dark for 72 h. The reaction was terminated by adding 2mercaptoethanol (10 mM final concentration), and the samples were dialyzed against 10 mM Tris-HCl, pH 8.0, and lyophilized. The samples were resuspended in stop solution, boiled for 3 min, and run on a linear 7.5-15% polyacrylamide gel according to the procedure of Laemmli (30). The unfixed gels were analyzed under fluorescent light (Chromato-YUB transilluminator model O-61, Ultraviolet Products, Inc., San Gabriel, CA) and then fixed and stained with Coomassie Brilliant Blue. The following proteins were used as molecular weight standards: phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), and a-lactalbumin (14,000). Fluorescence

Spectroscopy

Measurements

Technical steady-state fluorescence excitation and emission spectra were obtained with a Perkin-Elmer MPF-44A spectrophotofluorometer using excitation and emission band widths of 4 nm each. The steady-state emission anisotropy was determined using either a Polacoat dichroic polarizer (Polaroid Corp., Cambridge, MA) or a linear polarizer Polaroid HNP’B installed in the excitation path and a Polaroid HNP’B filter in the emission path. The relative intensities for the four combinations of vertically (v) and horizontally (h) polarized beams (I,, Zvb Zhh, Ii,“) were recorded in the “ratio mode.” Steady-state emission anisotropy was calculated as follows

GI”” - I”h ( r ) = GI,,

+ ZZ”,

where G = Zhs/Zhv is the grating correction factor introduced to normalize for the different sensitivity of the system to detect the horizontally and vertically polarized emission (31, 32). Fluorescence quenching of synapsin I was carried out using acryl-

Phosphorylation-induced

12586

Conformational

amide as a quencher. Protein samples at increasing concentrations of the quencher were prepared by adding small aliquots of a concentrated acrylamide solution (8M). When required, corrections were introduced according to Parker (33). The steady-state fluorescence data were analyzed according to the Stern-Volmer equation (34), F,/F

= 1 + K,.

[Q]

= TO/~

(2)

where F, and F are the fluorescence intensities measured in the absence and the presence of the quencher, K., is the Stern-Volmer constant, and [Q] is the quencher concentration. K,, is related to the bimolecular quenching constant (K,) and to the mean fluorescence lifetime in the absence of quencher (rO) as follows, K,

= Kq.7”

(3)

where rC, represents a measure of the accessibility of the fluorophore to the external environment. Nanosecond time-resolved fluorescence measurements were obtained using a time-correlated single photon counter (35-37) equipped with a thyratron-gated nitrogen flash lamp (model F199, Edinburgh Instruments, Edinburgh, Scotland) as a light source. The electronic modules were from Ortec (Oak Ridge, TN), the photomultiplier (model XP2020Q) was from Philips, and the multichannel analyzer (model BS27N) from Silena (Milano, Italy). The decay of the total fluorescence intensity was recorded under “magic angle” conditions (38), and the wavelength-dependent time shift of the photomultiplier (39) was determined in a separate experiment using NATA and anthracene as standards. Experimental curves, collected to resolve the spectra associated with the individual decay constants (decayassociated spectra, DAS) (40), were obtained by stepping the emission monochromator in increments of 5 nm between 310 and 400 nm, with a dwell time of 3 min on each sample position and 1 min on the lamp position. The energy transfer efficiency, ET, was obtained following the relationships developed by Eisinger (41) and Schiller (42) to calculate the efficiency of transfer over a distance R, ET =

U&/R ((Ro/R16

Y =- (TOD - TD) + 1) TB

Data

Analysis

Fluorescence Intensity Decay-The decay data were analyzed by nonlinear least squares method (44-46), and decay curves collected at multiple emission wavelengths were simultaneously analyzed according to the global procedure described by Knutson et al. (47). The experimental data were analyzed assuming that the fluorescence decay follows a multiexponential law, I(t)

= Z: a,.e-‘/‘~

(5)

where the relative amplitudes o(, and the decay constants 71 are the numerical parameters to be recovered. The best fit between the theoretical curve and the data was evaluated from the plot of residuals, the autocorrelation function of the residuals, and the reduced chi-square (x2) (46). The DAS were obtained by the global procedure (48) from decay curves collected every 5 nm for the same amount of time (see above). The decay of the fluorescence intensity as a function of the emission wavelength is given by I(t,X)

= LY~(A,).~-“‘~

+ an(h,).e-“‘2

+ . . . a,(A,).e-“‘n.

length-dependent parameters (the preexponential terms). A twodimensional plot of the preexponential terms uersus wavelength gives the spectra associated with each lifetime. In the DAS reported in the figures, fluorescence relative intensities at the various wavelengths were expressed as o(, .7, products. Fluorescence Anisotropy Decay-Equation 1 can be rewritten to represent the time dependence of the emission anisotropy as follows,

GL”(t) - L(t) r(f)

(‘3

According to this model, lifetimes do not depend on the emission wavelength, and the simultaneous analysis of all the curves provides n wavelength-independent parameters (the lifetimes) and n.j wave-

= GIJf)

+ BZ”h(L)

where (L(t) + 21-t,(t)) (also indicated elsewhere as s(t)) represents the decay of the total fluorescence intensity and does not depend on molecular reorientation whereas the term (ZJt) - I”h(t)) contains all the information on the rotational relaxation kinetics. A general expression relates the decay of the fluorescence anisotropy, r(t), to the correlation function for the dipole reorientation angle, 0 (49) F(f)

=

2/5

< < Pz [,iis(o)~~e(f)]

(8)

> >

where cP,>

= 312 -

l/2

is the second rank order parameter, and ,L, and b. are the unit vectors pointing along the absorption and emission dipoles at time 0 and time t, respectively. In many cases, the expression for can be approximated to a monoexponential decay function, and the anisotropy decay can be described by a sum of discrete exponential terms as follows (50, 51), r(t)

= 2 &.e-““I

(9)

where the sum of the preexponential terms p, is the anisotropy in the absence of rotation rO, and the & values are the rotational correlation times. The parameters for the decay of anisotropy r(f) were recovered from the analysis of the experimental decays of the polarization components I,,(t) and Idt) by the system analysis approach introduced by Gilbert (52). According to this method, the fitting functions to obtain r(f) are the following (53, 54) Cl,(t)

where R. is the “Forster critical distance” for 50% transfer efficiency, T$ is the lifetime of the donor including all nonradiative processes except energy transfer, and 7n is the lifetime in the presence of the acceptor. The decay of the emission anisotropy of synapsin I was measured as described previously (43), using a combination of two linear polarizers Polaroid HNP’B parallel (vv) and crossed (vh) with respect to the excitation and the emission paths. Decay curves for the polarized components of the emitted fluorescence were collected separately within the same experimental time course by alternatively rotating the cell holder in the three positions, “lamp,” “I,,,” and “I+” Dwell times of 3 min were used for the samples (1, and Ivh) and 1 min for the excitation response function (lamp). The scaling factor G was obtained as described by Badea and Brand (38). Fluorescence

Changes of Synapsin 1

L(t)

= 1/3

S(t).(l

= l/3

s(t).0

+ SF(t))

(10)

- r(f)).

(11)

Common parameters were linked, and n/T terms were introduced in the analysis of anisotropy decay curves obtained at multiple temperatures. When indicated, anisotropy decay analysis was also performed using the method of the “sum and difference” (43). It has been shown that ellipsoids of revolution contain three exponential terms (51). However, in the simplest case of spherical rotors, a single correlation time can be related to the hydrated volume of the rotating molecule, V, by the Stokes-Einstein equation $J -

= (V.7)

1/(6D)

(12)

/ (k.T)

where n is the solvent viscosity, k is the Boltzmann constant, T is the absolute temperature, and D is the rotational diffusion coefficient. The variability of the decay parameters was evaluated by determining the joint con6dence intervals (55). All the steady-state and time-resolved fluorescence experiments were run at least twice using different preparations of synapsin I phosphorylated and/or labeled with extrinsic fluorescent probes. The interexperimental variability was less than 10%. RESULTS Steady-state shown in Fig. the hydrophobic

Intrinsic 1, synapsin head

Fluorescence of Synapsin I-As I contains 4 tryptophan residues in region (9). Steady-state fluorescence

spectra have been recorded 295

nm

in the

indole

by exciting

absorption

band.

the protein In

Fig.

samples at

2, the

steady-

state excitation and emission spectra of native synapsin I are presented together with the emission spectra of denatured synapsin I and NATA, a model compound for the emission of tryptophans totally exposed to the solvent. Under native conditions, the maximum of the emission intensity of synapsin I was centered at 330 nm, significantly blue shifted when compared with the emission spectrum of NATA. The spectral data suggest that tryptophan residues in synapsin I are deeply

Phosphorylation-induced NTCECLEAVED N-TERMINAL I-

2

Pl NH2f

p2

w

SHSH ww

w

SH w

SHSH ww

F2 y3

p3 mcoon

El COOH TAIL REGION

HEAD REGION

FIG. 1. Structural model of synapsin I. The hydrophobic, collagenase-resistant head region and the basic, collagenase-sensitive tail region together with the three phosphorylation sites PI, P2, and P:, (Ser.9, Ser-566, and Ser-603) are indicated for synapsins Ia and Ib (upper and lower peptides, respectively). The two polypeptides are virtually identical with the exception of a small COOH-terminal region (dashed areas). Synapsin I contains 3 cysteine (Cys-223, Cys360, Cys-370) and 4 tryptophan (Trp-126, Trp-227, Trp-335, Trp356) residues that are restrict,ed to the head region. Their location is shown in the figure (-SH, cysteine; W, tryptophan). In order to assess the location of pyrene and acrylodan derivatization, synapsin I was cleaved at the free cysteine sites by NTCB. The three synapsin I-spanning fragments (29.kDa NH1-terminal fragment, 15-kDa middle fragment, and 35/39kDa COOH-terminal fragment) generated by this procedure are shown in the top of the figure. Two additional fragments (40-kDa NHZ-terminal/middle fragment and U/54-kDa middle/COOH-terminal fragment) are also produced from incomplete cleavage. For further explanations, see “Experimental Procedures” and Bahler et al. (8).

-2 5 20.0

z

150

B 2 u” E z: P 2 2

Excitation

Ermssmn

j

100

5.0

0.0 220

260

300 340 360 WAVELENGTH (nm)

420

460

FIG. 2. Steady-state fluorescence excitation and emission spectra of synapsin I. Synapsin I (0.2 mg/ml) was dissolved in 10 mM Tris-HCI, pH 7.4, 30 mM NaCl, 0.1 IIIM EDTA, 0.1 mM DTT in the absence (solid line) or presence of 6 M guanidine HCl (dot-dashed line). The excitation wavelength was 295 nm, and the relative fluorescence intensity is reported in arbitrary units (A.U.). The emission spectrum of NATA is also reported for comparison (dashed line). The spectra were normalized to the maximum of the fluorescence intensity of native synapsin I. inside the hydrophobic head region. When synapsin I is exposed to denaturing conditions (6 M guanidine HCl), the emission spectrum closely traces the fluorescence profile of NATA, consistent with a complete exposure of the tryptophan residues to the external environment during the unfolding process. The chemical environment of the intrinsic fluorophores of synapsin I was investigated by fluorescence quenching techniques. Based on this method, the accessibility of tryptophan residues is evaluated by their ability to interact with a suitable quencher dissolved in the assay buffer. Acrylamide, a polar and uncharged compound, was used as a quencher. The quenching of synapsin I fluorescence was studied at low (0 mM NaCl) and high (200 mM NaCl) ionic strength conditions. Linear Stern-Volmer plots were obtained from fluorescence intensity measurements (Fig. 3), and Ksy values of 2.5 and 1.51 M-i were calculated in low and high salt, respectively. The corresponding quenching rate constants Kq were in the order of magnitude of those found for proteins containing

buried

12587

I

SH w

; 4

Changes of Synapsin I

FRAGMENTS C-TERMINAL

-I-l-

Pl NH

MIDDLE

Conformational

O.i.bOO

[Acrylamide]

M

FIG. 3. Stern-Volmer plot of acrylamide-induced quenching of synapsin I fluorescence. The quenching of the intrinsic fluorescence of dephosphorylated synapsin I induced by acrylamide was measured at the steady state using excitation and emission wavelengths of 295 and 335 nm, respectively. Synapsin I (0.2 mg/ml) was dissolved in 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM DTT in the absence (filled triangles) or the presence (open triangles) of 200 mM NaCl in the medium. The data were fitted using linear regression according to Equation 2. The points represent the mean (+ S.E.) of three separate experiments. The Stern-Volmer constants (KS,) were 2.50 M-l and 1.51 M-l at 0 and 200 mM NaCl, respectively.

unexposed tryptophan residues (56). However, the accessibility of the fluorophores to the polar quencher was higher in low salt conditions (K, = 0.48 x 10’ M-i s-l) than in high salt conditions (K, = 0.27 x 10’ M-’ s-l). No significant effects of phosphorylation were observed on both the steady-state emission spectrum and the quenching of synapsin I fluorescence (data not shown). Decay of the Intrinsic Fluorescence of Synapsin f-Timeresolved fluorescence studies on synapsin I were carried out at 10 “C under various experimental conditions (low and high ionic strength of the medium, denaturation, and site-specific phosphorylation). Decay curves were collected at three emission wavelengths (330, 350, and 370 nm) and were simultaneously analyzed by the global method. In Fig. 4 a typical result of the analysis of a single fluorescence decay curve is shown together with the statistical parameters used to judge the goodness of fit (plot of the residuals and plot of the autocorrelation function of the residuals). The decay constants obtained with the dephosphorylated form of synapsin I in the presence of 0 mM NaCl, 200 mM NaCl, or 6 M guanidine HCl and those obtained with the fully phosphorylated form of synapsin I are presented in Table I. The average fluorescence lifetime, CT>, increased from 5.21 to 5.61 ns with increasing ionic strength and dropped to 3.97 ns under denaturing conditions. In all cases, the decay of the fluorescence intensity was best described by a sum of three discrete exponential terms. The three decay components were found to be 0.2, 3.0, and 7.0 ns, respectively, and were not significantly affected by the ionic strength, phosphorylation or denaturation. Although in the latter case an increase of the shortest lifetime was observed (see Table I), under our experimental conditions this result could be an effect of the covariance between the recovered parameters. On the other hand, the amplitudes associated with each lifetime were significantly changed by the ionic strength and denaturation, as expected from the changes of the average lifetimes CT>. The values of the preexponential terms recovered as a function of the wavelength after normalization to the relative steady-state emission spectrum are presented in Fig. 5. Under native conditions and low ionic strength (Fig. 5, upperpanel), the maximum of the fluorescence intensity was centered around 330 nm (see also Fig. 2) with a major contribution of the long lifetime component (55-60% of the total steady-state

Phosphorylation-induced

1:,-b 9B-

FIG. 4. Analysis apsin I. Fluorescence

Channel

#

Channel

#

Conformational

L r

of the fluorescence

intensity

decay of syn-

decay data were obtained at 10 “C using an excitation wavelength of 295 nm (band pass 4 nm), and the fluorescence emission was observed at 330 nm (band pass 10 nm). The protein concentration was 0.2 mg/ml in a buffer containing 10 mM Tris-HCl, pH 7.4, 200 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT. The calibration time for each channel was 0.0862 ns. Photon counts are reported on the y axis in a linear scale. The excitation lamp profile is indicated as L. The solid noise-free curves represent the theoretical parameters convolved with the lamp. In a, the results of the analysis are shown for a biexponential decay function; in b, the same decay data were analyzed as three exponential components. The plot of the weighted residuals (center-graphs) and autocorrelation of the weighted residuals (upper right inset) are shown. The fluorescence decay parameters were: 01~ = 0.65, r1 = 0.54 ns, CQ = 0.35, r2 = 5.89 ns, x2 = 2.27 for a biexponential decay (a) and O(I = 0.78, TI = 0.13 ns, a12 = 0.11, T? = 3.22 ns, N:, = 0.11, T:~ = 6.81 ns, x’ = 1.14 for three fluorescence decay components (b).

fluorescence at 330 nm) and a minor one of the intermediate lifetime component (35-40%), the short lifetime component contributing less than 10%. At higher ionic strength (Fig. 5, middle panel), the contribution of the long lifetime component increased to 65-70% of the total fluorescence intensity, consistent with the changes of the mean lifetime. After exposure of synapsin I to 6 M guanidine HCl (Fig. 5, lower panel), the maximum of the fluorescence intensity was shifted to 350 nm with a decrease of the total emission to about 40% of the fluorescence observed with the native protein. In addition, the relative contribution of the 3.0-ns lifetime component became predominant (54%) whereas the long lifetime com-

Changes of Synapsin I

ponent decreased its fractional contribution to 38%. Decay-associated Spectra-The use of spectral resolution in the nanosecond time domain to unravel conformational changes of proteins has been reported (40,57, 58). In view of the wavelength dependence of the preexponential terms describing the fluorescence decay of synapsin I and their changes in response to ionic strength and denaturation, it was of interest to resolve the decay-associated spectra for the various phosphoforms of synapsin I. The DAS obtained from the dephosphorylated and the three phosphorylated forms of synapsin I are shown in Fig. 6. With dephosphorylated synapsin I (Fig. 6, top-left panel), the spectra associated with the intermediate and the long lifetimes were centered at about 330 nm and provided the largest contribution to the total fluorescence whereas the maximum of the short lifetimeassociated spectrum appeared to be located in a lower frequency region (X,,, at approximately 340 nm) and accounted for a very low percentage of the total fluorescence intensity. When synapsin I was phosphorylated on the head site by CAMP-dependent protein kinase (site 1; Fig. 6, top-right panel), the spectrum associated with the long lifetime, although less prominent, was still centered at 330 nm; however, the spectrum associated with the intermediate lifetime was slightly red shifted (X,,, at 335 nm), and the spectrum associated with the short lifetime became more prominent in the blue region, with a broad peak around 320 nm. Synapsin I phosphorylated on the tail sites by Ca’+/calmodulin-dependent protein kinase II (sites 2 and 3; Fig. 6, bottom-left panel) and the fully phosphorylated form of synapsin I (sites 1, 2, and 3; Fig. 6, bottom-right panel) exhibited a blue shift of the spectrum associated with the short lifetime accompanied by a red shift affecting the spectra associated with both the intermediate and long lifetimes (with peaks at 340 nm in the 2,3-phosphoform and at 335 nm in the fully phosphorylated form). Although the phosphorylation-dependent changes in the distribution of the preexponential terms were observed in the absence of significant variations of the individual lifetimes and of the static emission spectra, they were consistent with the change in the average lifetime reported in Table I. Characterization of the N-(I-Pyrenyljmaleimide and Acrylodan Adducts of Synapsin I-Synapsin I contains 3 cysteine residues (Cys-223, Cys-360, Cys-370) located exclusively in the head region (8, 9). Two specific thiol-reactive fluorescent probes were coupled to synapsin I: acrylodan, a reagent characterized by a 6-acryloyl-2-dimethylaminonaphthalene moiety very sensitive to solvent polarity (27), and N-(lpyrenyl)maleimide, a slow decaying fluorophore useful for analyzing the fluorescence anisotropy decay of large proteins (25). Using a 5-fold molar excess of the reagents over cysteine residues, the stoichiometry of incorporated label was between 0.68 and 0.87 mol of pyrene chromophore and between 0.40 and 0.48 mol of acrylodan per mol of synapsin I. The fluorescent synapsin I conjugates were tested for binding to synaptic vesicles and F-actin and behaved similarly to native synapsin I in terms of both binding constants and effect of phosphorylation (data not shown). In order to identify the derivatized cysteine residues, a cysteine-specific chemical cleavage of synapsin I adducts was carried out using NTCB (8). Since the digestion reaction consists in the S-cyanylation of cysteine residues bearing free sulfhydryl groups followed by cleavage of the peptide bond at the P-thiocyanoalanine (59), an abnormal fragment pattern should result if a particular cysteine residue is preferentially derivatized by the fluorescent reagent. In addition, the analysis of the residual fluorescence on the fragments originating from incomplete cysteine cleavage could provide further in-

Phosphorylation-induced

Conformational

Lifetimes (7) of the time-resolued decay of synapsin I was measured

Changes of Synapsin I

12589

TABLE I fluorescence components of synapsin I at the emission wavelengths of 330, 350,

The fluorescence and 370 nm, and the data were analyzed by global analysis with the decay times linked across the emission band. The average lifetime is defined as S(a,. T:)/.Z( 01y.7~).The error limits refer to the 67% joint confidence interval (55).

Dephosphorylated (0 mM NaCl) Dephosphorylated (200 mM NaCl) Dephosphorylated (6 M guanidine) Phosphorylated (sites 1, 2, 3) (0 mM NaCl)

330

360

420

390

WAVELENGTH

(nm)

WAVELENGTH

(nm)

WAVELENGTH

FIG. 5. Relative cence decay denaturation.

0.21 0.16 0.62 0.16

amplitudes of the of synapsin I in response

450

(nm)

time-resolved to ionic

strength

fluoresand

The fluorescence decay of dephosphorylated synapsin I (0.2 mg/ml) was measured at 10 “C in a buffer containing 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM DTT, with no addition (upper panel), after addition of 200 mM NaCl (middle panel), and after

addition

wavelength

of 6

M

guanidine

HCl

(lower

panel),

using

an excitation

of 295 nm. The results refer to a global analysis run

simultaneously on three decay curves collected at emission wavelengths of 330, 350, and 370 nm for each set of experimental conditions. The data, plotted as the product OI,.T~/&.T~, were normalized to the relative steady-state emission spectrum (solid line in thegraph). The error bars refer to the 67% joint confidence intervals. The following symbols are used: filled circles, short decay time (rl); open triangles, intermediate decay time (~2); filled triangles, long decay time (TR).

formation on the location of the fluorescent probes. The results of such an experiment are reported in Fig. 7. Compared with underivatized synapsin I, the amounts of NH,-terminal, middle, and middle/COOH-terminal fragments originating from N-( 1 -pyrenyl)maleimide-derivatized synapsin I showed a net decrease whereas a compensatory

* + + c

0.05 0.05 0.15 0.02

2.86 2.76 3.04 2.62

ns k k + +

0.21 0.35 0.11 0.19

7.10 6.93 7.33 6.83

* f -c +

0.20 0.25 0.35 0.26

5.21 5.61 3.97 4.25

increase of undigested synapsin I took place. Moreover, the NH,-terminal/middle fragment appeared to be the only fluorescent peptide other than holosynapsin I. These observations indicate strongly that the N-(I-pyrenyl)maleimide adduct of synapsin I preferentially involves the residue Cys-223, which connects the NHP-terminal fragment to the middle fragment. The protein stain of the fragments generated by NTCB cleavage of acrylodan-synapsin I did not show major changes with respect to unlabeled synapsin I, and all the fragments were moderately fluorescent with the exception of the NH,terminal one (data not shown). This suggested that either the labeling with acrylodan was not as site specific as the one with N-(I-pyrenyl)maleimide or that it affected 1 of the 2 adjacent cysteine residues (Cys-360 or Cys-370) without inducing gross alterations of the fragment pattern on SDSpolyacrylamide gel electrophoresis. The steady-state fluorescence emission spectrum of acrylodan-derivatized synapsin I under native and denaturing conditions is shown in Fig. 8. The maximum of the emission intensity of native acrylodan-synapsin I was observed at 460 nm, representing one of the most blue shifted spectra reported for acrylodan-derivatized proteins (27). Transition from the native to the unfolded state following denaturation in 6 M guanidine HCl was accompanied by a marked red shift of the spectrum, with a peak at approximately 520 nm, suggesting a complete exposure of the fluorophore to the solvent. The absorption and emission spectra of the N-(l-pyrenyl)maleimide adduct of synapsin I are shown in Fig. 9. The emission spectrum of the pyrene-labeled protein showed three peaks at 376, 396, and 418 nm as expected for a pyrene derivatization with closed succinimido ring (26). Since intramolecular aminolysis has been reported for this kind of product (60), attention was paid to the appearance of the peak at 386 nm associated with the opening of the succinimido ring. In all the experiments performed, the pyrenyl-synapsin I adduct appeared to be substantially stable; aminolysis was detected only with samples kept for more than 4 days at room temperature (Fig. 9, lower panel). The fluorescence intensity decay of pyrenyl-synapsin I is shown in Fig. 10. Although only 1 cysteine residue/molecule of synapsin I appeared to be labeled by the reagent (see above), the fluorescence intensity of pyrenyl-synapsin I did not show a monoexponential decay. The global analysis performed on data collected with two time windows (0.086 and 0.169 ns/ channel) indicated that a three-exponential model provides an adequate description of the fluorescence decay. The three fluorescence decay components had lifetimes of 1.69, 12.81, and 60.46 ns, accounting for 3.5, 17.0, and 79.5% of the overall steady-state fluorescence emission. The fluorescence decay of the pyrenyl-synapsin I was also measured by exciting the sample in the tryptophan absorption band and by observing the emitted fluorescence from 330 to

Phosphorylation-induced

12590

Conformational

Changes of Synapsin I

WAVELENGTH

320

340

360

KAVELENCTH

360

400

420

(nm)

320

340

360

F1VELE9GTtl

(nm)

360

400

(nm)

FIG. 6. Decay-associated spectra of synapsin I in various states of phosphorylation. The fluorescence decay of dephosphorylated synapsm I (top-left panel), synapsin I phosphorylated on the head site by CAMPdependent protem kinase (site 1; top-rghtpanel), synapsin I phosphorylated on the tall sites by Ca’+/calmoduhndependent protein kinase II (sites 2 and 3; bottom-left panel), and synapsin I phosphorylated by both kinases (sites 1, 2, and 3; bottom-rrght pnnc>I) were measured at 10 “C m a buffer contamm g 10 mM Trls-HCl, pH 7.4, 30 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, using an excitation wavelength of 295 nm. The data, plotted as relative Intensity in each DAS, refer to the product (Y,.T,. Decay curves were collected every 5 nm, and the DAS were obtained by the global analysis method (see “Experimental Procedures”). In the figure, DAS data are shown every 10 nm for display purposes. Global reduced ,$ ranged between 1.15 and 1.46. The following symbols are used in the futures: ,Wed c1rtle.5, short decay time (T, = 0.2 ns); open trxmgles, medium decay time (rL = 3.0 ns); filled trianfiies, lo& decay time (i:, = 7.0 r&J. PYRENYL-SYNAPSIN I

SYNAPSIN I cs FL

94

-

67

-

43

-

cs

FL

SYNAPSIN I

~‘~;-:

MlDDLE C-TERMINAL N-TERMINAL M,DDlE C-TERMINAL

30 20

-

14

-

NTCB DIGESTION

N-TERMINAL

MIDDLE

-

+

-

+

FIG. 7. Cysteine-specific cleavage of synapsin I and of N(I-pyrenyl)maleimide-derivatized synapsin I. Unlabeled synapsin I and its N-(I-pyrenyl)maleimide adduct were subjected to cysteine specific cleavage with NTCB as described under “Experimental Procedures” and by Btihler et al. (8). Samples of the undigested and digested proteins (2 and 20 pg of protein, respectively) were separated by SDS-polyacrylamide gel electrophoresis with a 5-15X linear acrylamide gradient in the separating gel. In order to assess the relative ratios of the generated synapsin I fragments and whether they had been labeled by the fluorescent reagent, the unfixed gel was analyzed under UV light (FL) and subsequently stained with Coomassie Brilliant Blue (CS). For further details, see “Experimental Procedures” and Fig. 1.

370 nm. Under these conditions, the intermediate and long fluorescence lifetimes of tryptophan residues dropped from 7 to 4 ns and from 3 to 0.8 ns, respectively, consistent with the existence of a collisional quenching process due to fluorescence resonance energy transfer. The transfer efficiencies observed at the various emission wavelengths were calculated

360

430

460

630

660

2 6 30

WA”El,ENGTH (nm) FIG. 8. Steady-state fluorescence emission spectrum of acrylodan-derivatized synapsin I. Acrylodan-labeled synapsin I (0.06 ma/ml) was dissolved in 10 mM Tris-HCl, pH 7.4, 30 mM NaCl, 0.1 mM EDTA in the absence (solid line) or the presence (dashed line) of 6 M guanidine HCl. The excitation wavelength was 380 nm. The two spectra depicted in the figure are normalized to the peak intensity of the native labeled synapsin I. The contribution of the Raman peak is evident in the spectrum recorded under denaturing conditions. A.U., arbitrary units.

according to Equation 4 and are shown in Table II. The results are consistent with an excited state molecular interaction between the tryptophan residues and the pyrene moiety. Time-resolved Anisotropy Decay of Pyrenyl-synapsin I-In order to investigate whether the changes of the intrinsic fluorescence observed upon site-specific phosphorylation were accompanied by modifications of the hydrodynamic properties of synapsin I, the fluorescence anisotropy decay of the N-(lpyrenyl)maleimide adducts of the protein in the various phosphorylation states was measured. In Fig. 11, a typical set of experimental curves is shown together with the statistical evaluation of the fitting procedure. The parallel (Iv) and perpendicular (It,) polarized components of the fluorescence

Phosphorylation-induced

Conformational

Changes of Synapsin 1

6000

!

12591

Pt

WAVELENGTH(m)

4 10 0066 ns

:

:

20

30

I 40 Time

:

:

:

;

50 (ns)

60

70

60

400

500 0

90

WAVELENGTH(m)

FIG. 9. Absorption spectrum and fluorescence emission spectrum of synapsin I-N-(1-pyrenyl)maleimide conjugates. Upper panel, unlabeled synapsin I (dashed line) and N-(l-pyrenyl)maleimide-labeled synapsin I (solid line) were dissolved in 25 mM Tris-HCl, pH 8.0, 200 mM NaCI, 1 mM EDTA at a concentration of 0.55 mg/ml. Absorbance was recorded at wavelengths ranging from 220 to 450 nm. Synapsin I concentration was determined by the method of Lowry et al. (28) using a calibrated synapsin I solution as a standard. The stoichiometry of labeling was estimated to be about 0.87 mol of pyrene chromophore per mol of synapsin I. Lower panel, steady-state fluorescence emission spectrum of the N-(l-pyrenyl)maleimide adduct of synapsin I (0.05 mg/ml) dissolved in a buffer containing 10 mM Tris-HCl, pH 7.4, 30 mM NaCl, 0.1 mM EDTA at 6 “C (solid line). The excitation wavelength was 337 nm with excitation and emission band widths of 4 nm. The emission spectrum of the same sample kept at room temperature for more than 4 days showed that intramolecular aminolysis had occurred (dashed line). A. U., arbitrary units.

decay were collected at 5, 12, and 22 “C, and the data were analyzed by the global method. Since it was not possible to recover complex rotational relaxations from the experimental data, the decay of fluorescence anisotropy was analyzed assuming a spherical symmetry. Monoexponential fitting functions were obtained for all the forms of synapsin I at all the tested temperatures. The experimentally determined correlation times are shown in Table III. The values of the decay constants found for dephosphorylated synapsin I (415 ns at 5 ‘C!) and for synapsin I phosphorylated at site 1 (head region) by CAMP-dependent protein kinase (370 ns at 5 “C) are much higher than might be expected for a spherical protein whose molecular weight is around 80,000. Closely similar decay constants were obtained in the presence of 1 mM DTT (data not shown) or at higher ionic strength conditions (200 mM NaCl, Table III). A Stokes radius of 60-63 A was calculated from the Einstein-Stokes relation for dephosphosynapsin I in good agreement with the results obtained previously by gel filtration chromatography (6). However, the statistical parameters obtained from these experiments were not satisfactory, the x2 ranging between 1.6 and 2.0. Interestingly, the anisotropy decay of pyrenyl-synapsin I phosphorylated at sites 2 and 3 (tail region) by Ca’+/calmodulin-dependent protein kinase II or at all three sites exhibited a 3-4-fold shorter correlation time (120-i30 ns at 5 “C), corresponding to a Stokes radius of 41-43 A (Fig. 12). This parameter is close to the expected value for a spherical protein of 80,000 molecular weight, and the recovered statistics reflected the goodness of

0

100

200 300 Channel#

LOO 200 Channel/2

FIG. 10. Analysis of the fluorescence intensity decay of N(1-pyrenyl)maleimide-derivatized synapsin I. The fluorescence of pyrenyl-synapsin I was measured at 10 “C using excitation and emission wavelengths of 337 and 396 nm, respectively. The protein concentration was 0.05 mg/ml in a buffer containing 10 mM TrisHCl, pH 7.4, 30 mM NaCl, 0.1 mM EDTA. Photon counts were collected at two channel calibration times (0.086 and 0.169 ns/ channel) in order to achieve a good resolution in a wide lifetime range. In the upper panel, the decay data convolved with the lamp and the resulting fitted decay curves obtained by the global analysis with linkage of the decay times are reported. The best tit was for a three-exponential decay with the following resulting parameters: a1 = 0.44, TV = 1.69 ns, cyz = 0.28, T* = 12.81 ns, (Ye = 0.28, r3 = 60.46 ns, global reduced x2 = 1.20. In the lower panel, the respective plots of the weighted residuals and of their autocorrelation are shown. TABLE II pyrene-tryptophan energy transfer efficiency for dephosphorylated pyrenyl-synapsin I The experimental conditions used were the same as described in the legend to Fig. 10. The mean lifetimes in the presence of the acceptor, CT+, were calculated as Z(a,.r1)/Z(ai.r,). The transfer efficiency, ET, was calculated according to Equation 4. In the absence of a detailed knowledge of the secondary and tertiary structure of synapsin I, it was not possible to obtain an accurate estimate of the critical distance R. for this donor-acceptor pair. Excitation at 295 nm. Intramolecular

Emission wavelength



nm

Il.3

340 350 360

1.80 2.25 2.79

ET

0.66 0.57 0.47

the fitting (x” = 1.19-1.22; see Fig. 11). The time 0 anisotropy values recovered from the various forms of pyrenyl-synapsin I were not significantly different and ranged between 0.13 and 0.16. When the anisotropy decay of the intrinsic fluorescence was analyzed using a monoexponential decay model, correlation times in agreement with those determined from pyrene fluorescence were obtained but with higher time 0 anisotropy values (data not shown). The higher value obtained for the latter parameter (r. = 0.23) may reflect a more rigid environment of the tryptophan residues. In order to overcome the difficulties in resolving complex

12592

Phosphorylation-induced

Channel

Conformational

Changes of Synapsin I

TABLE III Anisotropy decay parameters of pyrenyl-synapsin I Emission anisotropy decay of N-(l-pyrenyl)maleimide-derivatized synapsin I in various phosphorylation states was analyzed as described under “Experimental Procedures.” The various forms of derivatized synapsin I were dissolved in a buffer containing 10 mM TrisHCl, pH 7.4, 30 mM NaCl, 0.1 mM EDTA at a concentration of 0.05 mg/ml and analyzed at different temperatures. The inclusion of reducing agents (1 mM DTT) did not change the anisotropy decay parameters. The anisotropy decay is expressed as: r(t) = pee-““, where fl represents the time 0 anisotropy and 4, the correlation time. Stokes radius (SR) was calculated following the Stokes-Einstein equation for spherical molecules (see “Experimental Procedures.“)

#

Synapsin

I

Temperature “C 5 12 22

Dephosphorylated

1)

100

ZOO

300

Channel

100

#

500

0

100

FIG. 11. Analysis of the time-resolved anisotropy decay of N-(1-pyrenyl)maleimide-derivatized synapsin I. The decays of the polarized components Iv and In of the fluorescence intensity are shown for a typical experiment obtained with N-(l-pyrenyl)maleimide-derivatized synapsin I phosphorylated by Ca’+/calmodulin-dependent protein kinase II (upper panel). The protein was dissolved at a concentration of 0.05 mg/ml in 10 mM Tris-HCl, pH 7.4, 30 mM NaCl, 0.1 mM EDTA. For display purposes, only the curves collected at the temperatures of 5 and 12 “C are shown. The excitation and emission wavelengths were 337 and 396 nm, respectively. The statistical parameters (weighted residuals and autocorrel&on functions) obtained by the global analysis are reported in the lower panel. The anisotropy decay parameters obtained at 5 “C were: OL, = 0.17, ri = 1.37 ns, 0~~ = 0.095, 7* = 9.34 ns, o/~ = 0.054, 73 = 51.4 ns, 0 = 0.152, 4 = 121 ns with global reduced x2 = 1.198 and 1.203 for the fluorescence intensity and anisotropy decays, respectively.

rotational behaviors of the protein as a whole (as might be the case for dephosphorylated synapsin I), the global approach was designed to take advantage of both the short tryptophan fluorescence lifetime and the longer fluorescence time course of pyrene. Global analysis was run simultaneously on the intrinsic and the extrinsic fluorescence anisotropy decay data, with linked correlation times and free floating p terms. Even this kind of analysis failed to resolve multiple decay components that may be related to the rotational diffusion around a major and a minor axes of symmetry. However, a decrease in x2 was observed when a fixed correlation time of 1.5-2.0 ns was introduced into a biexponential decay model. Since the statistical improvement was mainly provided by the intrinsic fluorescence anisotropy decay data (& = 0.220, & = 304 ns, pZ = 0.014, & = 1.5 ns at 12 “C), it is tempting to assign the additional short correlation time to the independent flexibility of the indole ring. DISCUSSION

Proteins have a large potential for structural variations, which are advantageous for transmitting allosteric information between nonadjacent domains. In this paper, the steadystate and time-resolved fluorescence of synapsin I, due to tryptophan residues or to extrinsic covalently attached fluorescent probes, have been characterized in various ionic strength conditions and phosphorylation states. This study was carried out with the purpose of better understanding the effects of the microenvironment on synapsin I physical prop-

SR

0.14 0.14 0.15

A 63.0 63.0 63.1

5 12 22

0.13 0.14 0.13

370 294 220

60.6 60.7 60.7

(site

Phosphorylated

(sites

2 and 3)

5 12 22

0.15 0.16 0.15

121 96 72

41.8 41.8 41.9

Phosphorylated

(sites

1, 2, 3)

5 12 22

0.13 0.13 0.14

128 105 74

42.7 43.0 42.3

5 12 22

0.13 0.14 0.14

398 300 224

61.1 61.2 61.1

200

Channel/Z

4 Il.3 415 329 246

Phosphorylated

\ 0

fl

Dephosphorylated

(200

mM)

NaCl

I 0

100

200 Channels

300

400

500

#

FIG. 12. Effect of the phosphorylation state of N-(l-pyrenyl)maleimide-derivatized synapsin I on the decay of fluorescence anisotropy. Comparison of the anisotropy decay data obtained with dephosphorylated synapsin I (noisy curue) and with synapsin I phosphorylated to near full stoichiometry by CAMPdependent protein kinase and Ca’+/calmodulin-dependent protein kinase II (dots) at 12 “C. Both forms of synapsin I had been conjugated with N-(I-pyrenyl)maleimide prior to the phosphorylation reaction as described under “Experimental Procedures.” The proteins were dissolved at a concentration of 0.05 mg/ml in 10 mM Tris-HCl, pH 7.4, 30 mM NaCl, 0.1 mM EDTA. The decay data, plotted on the y axis in a logarithmic scale, refer to the difference Z,(t) - Z”h(t). The noise-free solid curues represent the fitting functions obtained from the analysis of the data using the method of the sum and difference (43).

erties and hydrodynamics. Although steady-state fluorescence measurements provide an intensity and time-averaged description of the underlying decay processes, nanosecond timeresolved fluorescence spectroscopy is able to monitor relevant biological events occurring in this time domain such as rotational movements of the whole protein or rapid conformational rearrangements in response to environmental stimuli (24. The relationship between the primary sequence and the arrangement of a folded, biologically active conformation is

Phosphorylation-induced

Conformational

of paramount importance in protein chemistry. In agreement with the structural predictions made from the amino acid sequence (9), the data presented here demonstrate that under native conditions, tryptophan and cysteine residues located in the head region of synapsin I are surrounded by a hydrophobic environment and seem to be segregated from the external aqueous medium. In fact, the emission spectrum of the intrinsic fluorescence is markedly blue shifted in native conditions, and this effect is abolished by denaturation. Similar results were observed when synapsin I was derivatized with acrylodan, a sulfhydryl-specific fluorescent probe extremely sensitive to the dielectric environment (27); the emission peak found in the present work for this adduct is one of the most blue shifted that have ever been reported for acrylodan-derivatized proteins (27, 61). The weak quenching of the intrinsic fluorescence of synapsin I by the uncharged water-soluble acrylamide also supports the poor accessibility of the fluorophores. Hence, as observed for many proteins, the head region of synapsin I is likely to acquire and maintain a folded conformation by burying nonpolar residues in the interior. Within this three-dimensional arrangement, tryptophan and cysteine residues can come in close contact, as indicated by the occurrence of resonance energy transfer. The analysis of the decay of the intrinsic fluorescence of synapsin I revealed the presence of three decay components of 0.2,3.0, and 7.0 ns, with the intermediate and long lifetimes that give the predominant contribution to the total fluorescence intensity. In proteins containing multiple tryptophans (24), the real significance of the dynamic fluorescence components is still disputed. Only in few favorable cases (57), it has been possible to assign individual decay constants to unique tryptophan residues; in most cases even single tryptophan-containing proteins give rise to complex decay kinetics (24). In the case of synapsin I, containing 4 tryptophan residues and exhibiting a three-exponential decay behavior, no one-to-one assignment is possible, but conformational heterogeneity may provide a reasonable interpretation of these findings. According to this hypothesis, the absolute values of the decay constants together with the spectral distribution of their relative amplitude might reflect different excited state environments whereas pure changes of the amplitude contributions may be associated with transitions between different conformational states. Synapsin I steady-state emission spectrum and fluorescence lifetimes were not significantly affected under a series of experimental conditions involving various salt concentrations, various phosphorylation states, or denaturation, suggesting the existence of a very stable environment surrounding the tryptophan residues. However, the amplitudes associated with the lifetimes were found to be a more sensitive index of structural perturbations and were significantly affected by the environmental stimuli tested. The amplitude of the long lifetime component, which accounts for 55-60% of the total fluorescence intensity at low salt concentrations, was enhanced by increasing the ionic strength and markedly depressed upon denaturation. These data suggest that the increase in ionic strength of the medium is able to induce a conformational change that further decreases the exposure of the tryptophan(s) responsible for the long fluorescence decay component. This explanation, compatible with the existence in the head region of a tertiary structure strongly dependent upon hydrophobic interactions, is supported by the steadystate acrylamide quenching measurements, where the relatively low accessibility of the fluorophores observed in low ionic strength was decreased further by exposure of synapsin I to high salt concentrations.

Changes of Synapsin I

12593

Protein phosphorylation represents a mechanism of paramount importance by which extracellular signals can modulate intracellular events. It is generally assumed that phosphorylation can activate or inactivate specific substrate proteins by inducing transitions between different conformational states. Structural changes associated with phosphorylation have been reported for several proteins, including neurofilament proteins (62) and glycogen phosphorylase (63). The introduction of phosphate groups at sites 2 and 3 of synapsin I leads to decreased synaptic vesicle and Factin binding activities and virtually abolishes F-actin bundling (7, 19), suggesting that some critical conformational change of the synapsin I molecule might occur. However, surface activity analysis as well as CD spectroscopy have been unable to detect significant changes among the various states of phosphorylation of synapsin I.’ These observations suggest that any phosphorylation-induced structural transitions that do occur might be detected only with sensitive intramolecular probes. Two distinct approaches were followed in the present study, namely the analysis of the decay-associated spectra of tryptophan fluorescence and the fluorescence anisotropy decay of synapsin I labeled with the long lifetime fluorescent probe N-( l-pyrenyl)maleimide. The resolution of the decay-associated spectra of the intrinsic fluorescence of synapsin I revealed that in the absence of detectable changes in the steady-state emission spectrum, phosphorylation of synapsin I on the tail (sites 2 and 3) or on the tail and head (sites 1, 2, and 3) induces a red shift with decreased intensity of the spectra associated with the intermediate and long lifetimes and a blue shift with an increased intensity of the spectrum associated with the short lifetime. The decay-associated spectra observed with synapsin I phosphorylated on the head (site 1) were qualitatively similar to the other phosphoforms, with the exception that the spectrum associated with the long lifetime was not significantly red shifted. These effects suggest that a conformational change had occurred involving an increase in the polarity of the microenvironment surrounding the fluorophore(s) related to the intermediate and long lifetimes accompanied by an opposite effect on the fluorophore(s) associated with the short lifetime. Whether these changes reflect higher exposure to the solvent or internalization of the fluorophores cannot be determined at present. Although the fluorescence changes were relatively similar among the various phosphoforms, the effects were more marked when synapsin I was phosphorylated on the tail sites. Of particular interest, phosphorylation of the tail sites had an effect on the conformation of synapsin I which was detectable far beyond the immediate sequence around the phosphorylation sites (i.e. at the level of the tryptophan residues of the head region), proving that each domain of synapsin I is not independent but interacts with the other. Moreover, the selective involvement of synapsin I intrinsic fluorescence components under the experimental conditions tested further underlines that each class of tryptophan residues has peculiar photophysical properties. The time-resolved fluorescence of N-( 1-pyrenyl)maleimide conjugated to synapsin I exhibited three exponential decay components. The origin of this complex fluorescence decay is not clear since synapsin I appears to be derivatized by N-( lpyrenyl)maleimide only on Cys-223. However, a multiexponential decay of pyrene fluorescence when the reagent is bound to a single site of a protein is a very common finding (see, e.g. 64,65). The predominant contribution of the lifetime of 60 ns to the fluorescence emitted by pyrenyl-synapsin I ’ M. Ho, M. Bahler, A. J. Czernik, Kaiser, and P. Greengard, manuscript

W. Schiebler, F. J. Kezdy, in preparation.

E. T.

12594

Phosphorylation-induced

Conformational

provided an average lifetime suitable for studying the rotational motions of large proteins. The results obtained by studying the fluorescence anisotropy decay of pyrenyl-synapsin I revealed that dephosphorylated synapsin I under low ionic strength conditions had a fairly large correlation time, corresponding to a Stokes radius of approximately 60-63 A assuming a spherical symmetry. This value is in full agreement with the Stokes radius obtained by Ueda $nd Greengard (6) using gel filtration chromatography (59 A). This very high value for a protein of 80,00086,000 molecular weight might be attributed either to the marked asymmetry of synapsin I or to an oligomeric organization. The fact that the long correlation time did not change significantly in the presence of reducing agents or high salt concentrations seems to exclude that oligomerization due to formation of S-S bridges or hydrophobic interactions represents a dominant phenomenon at the synapsin I concentration tested (0.05 mg/ml). Moreover, gel filtration chromatography and sucrose density gradient centrifugation have demonstrated that synapsin I in diluted solution (below 0.2 mg/ ml) is predominantly in monomeric form whereas at higher concentrations (over 0.4 mg/ml) several oligomeric species are generated (6). Although in the case of synapsin I, the analysis in terms of a multiexponential anisotropy decay does not seem to lead to a straightforward interpretation of the data, the long correlation time found for the dephosphorylated and the head phosphorylated forms is consistent with a highly elongated shape of the molecule. This feature appears to be lost when the protein is phosphorylated on the tail region. In fact, phosphorylation of synapsin I on the tail sites induced a 3-4-fold decrease of the correlation iime with a change in the Stokes radius from 60-63 to 41-43 A. Such a change could be induced by a folding of the tail region toward the head region so that synapsin

I tends to assume a more spherical

symmetry.

However, it cannot be excluded that in conditions in which synapsin I oligomerization is favored, these conformational transitions might also affect the ability of synapsin I to selfassociate. In conclusion, the results reported here demonstrate that the introduction of two negative phosphate groups in the positively charged tail region triggers some major conformational changes affecting both the photophysics and the hydrodynamics of synapsin I. Whether these allosteric modulations are involved directly in the biological function of synapsin I within the nerve terminal and in the regulation of the interactions with synaptic vesicles and F-actin remains to be clarified but represents an attractive hypothesis. Acknowledgments-We wish to thank Drs. Ludwig Joseph M. Beechem, Dept. of Biology, The Johns Hopkins Baltimore, MD, for kindly providing the global analysis are also grateful to Drs. J. B. Alexander Ross, William Valtorta, and Andrew J. Czernik for helpful discussions reading of the manuscript.

Brand and University, routines. We Laws, Flavia and critical

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