ARCHIVES
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
Physicochemical Retinal Arrestin Susan
Kotake,*
BIOPHYSICS
15, pp. 126-133,199l
Patricia
Characterization Hey,?
Raghavendra
of Bovine
G. Mirmira,*
and Robert
A. Copeland**l
*The Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, Illinois 60637; and TDepartment of Biochemistry and Molecular Pharmacology, Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
Received
September
19, 1990, and in revised
form
October
29, 1990
intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed. o ISBI Academic press, L.
The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-‘, cm-’ using the tyrosinatetyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford calorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60°C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative
The visual transduction cascade serves as a structural and mechanistic paradigm for a large family of G-proteinmediated signal transducing systems (1). In this system, light absorption by the integral membrane protein rhodopsin (Rho)’ induces a conformational transition of Rho which results in its interacting with the heterotrimeric guanine nucleotide-binding regulatory protein, transdutin. The subunits of transducin then dissociate, leading to stimulation of the activity of the retinal cGMP phosphodiesterase (1). Light absorption by Rho also triggers its phosphorylation by a specific kinase, referred to as rhodopsin kinase (2). Recently another protein, known as arrestin, S-antigen, or 48-kDa protein, has been implicated in the negative control of visual transduction (3). This protein binds to phosphorylated Rho and interferes with the interaction of Rho with transducin (4). In this way the binding of arrestin to Rho prevents the transducin-mediated stimulation of the phosphodiesterase, and the system becomes desensitized. Cross-linking studies indicate that arrestin binds not only to Rho, but also to the retinal cGMP phosphodiesterase in the presence of ATP and light (5). Thus, arrestin-mediated densensitization may be the combined
’ To whom correspondence should be addressed at Department of Biochemistry & Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, Illinois 60637.
’ Abbreviations used: Rho, rhodopsin; NATEE, IV-acetyl-L-tryptophan ethyl ester; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide electrophoresis; Gdn-HCl, guanidinium hydrochloride.
126
0003-9%x/91
Copyright All rights
0 1991 by Academic
of reproduction
in any
form
gel $3.00
Press, Inc. reserved.
CHARACTERIZATION
result of interactions of the protein with the phosphodie&erase and Rho. Desensitization of agonist responsiveness is a common feature of G-protein-coupled receptor systems. Recently, Lefkowitz and co-workers reported the cloning and expression of an arrestin-like molecule from bovine brain which appears to interact with the P-adrenergic receptor system. This protein, termed @-arrestin, binds to agonistbound phosphorylated ,8-adrenergic receptor and attenuates the interaction of the P-adrenergic receptor with the G-protein G, (6). It thus appears likely that arrestinlike molecules play a major role in the desensitization of G-protein-coupled systems in general (7, 8). In addition to its role in signal transduction, arrestin also induces experimental autoimmune uveitis (9), a T-cell-mediated disease characterized by inflammation of the retina, uveal tract, and pineal gland (10). In an effort to understand the structural basis for signal transduction in G-protein-coupled systems, biophysical methods have been used to investigate conformational transitions of the proteins in these systems, and the interactions of these proteins with one another. The conformational changes accompanying the retinal-opsin interaction have been well characterized (11). More recently, rhodopsin-transducin interactions have been analyzed by fluorescence spectroscopy (12). The solution properties of retinal arrestin are of current interest not only because of the role of this protein in signal transduction, but also because of its high pathogenicity. The secondary structure of the bovine protein has been measured by circular dichroism and analyzed by computational methods (13). In addition, Fourier transform infrared spectroscopy has shown that the uveopathogenic octadecapeptide M from retinal arrestin forms macromolecular assemblies through intermolecular P-sheet formation under physiological conditions (14). In the present study, we report the characterization of bovine retinal arrestin by a number of spectroscopic methods. The complete amino acid sequence for this protein has recently been reported (13), and is reproduced in Fig. 1. Bovine arrestin contains 14 tyrosine residues and a single tryptophan residue within its primary structure (13). We therefore expected that the fluorescence spectrum of the protein would be dominated by tryptophan emission (15). Surprisingly, however, we find that the fluorescence of the single tryptophan is quenched in the native protein, resulting in a fluorescence spectrum which is dominated by tryosine emission. The observation of this tyrosine-like fluorescence spectrum appears to be correlated with the ability of arrestin to bind to lightadapted rod outer segments. Denaturation of arrestin by either thermal or chemical means significantly perturbs the fluorescence spectrum of the protein. These unusual fluorescence properties of bovine retinal arrestin may thus prove useful in following conformational transitions of this protein in solution.
OF
BOVINE
127
ARRESTIN
MKANKPAPNH
VIFKKISRDK
LVKGKRWVS
LTCAFRYGQE
TRLQESLIKK
SVTIYLGKRD DIDVMGLSFR
VIDHVERVEP
VDGVVLVDPE
RDLYFSQVQV
FPPVGASGAT
LGANTYPFLL
TFPDYLPCSV
MLQPAWDVG
AFATHSTDVE
EDKIPKKSSV
RLLIRKVQHA
PRDMGWPRA
KPLRlAVSLS
KEIWHGEPI
PVTVAVTNST
EKTVKKIKVL
VEQVTNVVLY
SSDWIKTVA
AEEAQEKVPP
NSSLTKTLTL
VPLLANNRER
RGIALDGKIK
HEDTNLASST
IIKEGIDKTV
VPFRLh4HPCP
EDPDTAKESF
MGILVSYQIK QDENFVFEEF
VKLTVSGLLG ARQNLKDAGE
KSCGVDFEIK EASVIMFFMSD
ELTSSEVATE YKEEKlDQEA
AMDE
FIG. 1. The complete amino acid sequence for bovine retinal arrestin highlighting the aromatic amino acids tyrosine (Y) and tryptophan (W). The sequence was taken from (3).
MATERIALS
AND
METHODS
N-Acetyl-L-tryptophan ethyl ester and N-acetyl-L-tyrosine were purchased from Sigma and used as received. All other reagents were the highest quality commercially available. Dark-adapted bovine retinas were purchased from George Hormel Co. (Austin, Minnesota) and stored, in the dark, at -80°C. until use. Arrestin was purified from the dark-adapted retinas as previously described (16). Rod outer segments were purified under dim red light conditions from dark-adapted retinas, as described by Hong and Hubbell (17). Under these conditions the resulting membranes are essentially devoid of endogenous arrestin, but retain rhodopsin and rhodopsin kinase activities (16). The rhodopsin concentration of the rod outer segments was determined spectrophotometrically at 498 nm using an extinction coefficient of 42.7 mM-‘, cm-’ (17). SDS-PAGE was performed on 16% acrylamide gels by the method of Laemmli (18). Protein bands on the gels were visualized either by Coomassie brilliant blue G-perchloric acid solution or silver staining. Protein concentration was determined calorimetrically by the method of Bradford (19) using either y-globulin or bovine serum albumin to obtain a standard curve. Fluorescence spectra were obtained with either a PTI alphascan or SLM 48000s spectrofluorometer. The samples were contained in a lcm quartz cuvette which was placed in a temperature-controlled sample holder. The temperature of the sample was maintained at 25’C throughout the experiments by circulating a water/ethylene glycol mixture through the sample holder from a refrigerated water bath. All of the reported spectra were obtained with the emission polarizer set at 54.75” to minimize spectral anomalies due to polarization bias and stray light scattering (20). Emission intensities were corrected for variation in instrument response by reference to a 3 g/liter solution of rhodamine B in ethylene glycol (20). The reported spectra have not been corrected for inner filter effects, but in all cases the absorptivity of the sample was kept below 0.1 to minimize such effects. The relative quantum yields of arrestin tyrosine and tryptophan fluorescence, reported in Table I, are referenced to the 354-nm emission band of a 5 pM solution of N-acetyl-L-tryptophan ethyl ester (NATEE) in 10 mM Tris-HCI buffer, pH 8.0, excited with 280-nm light. The NATEE solution was prepared by dilution of a 100 pM stock solution whose concentration was determined spectrophotometrically using an extinction coefficient of 5,600 M-km-’ at 280 nm (21). The quantum yield, 9, of a fluorophore can be related to the observed fluorescence intensity as follows (22):
F = Z,,(2.3 cd) a, where F is the fluorescence intensity, Z0 is the incident light intensity, e is the molar extinction coefficient for the fluorophore at the excitation
111
128
KOTAKE
wavelength, c is the sample concentration, and d is the pathlength of the cuvette. Note that the product ccd is equivalent to the absorbance of the sample at the excitation wavelength. If the quantities I0 and d are held constant, the quantum yield of any analyte can be determined by measuring its fluorescence spectrum along with the spectrum of a standard of known quantum yield, and using equation (22)
where a,, F,, and A, are the quantum yield, fluorescence intensity, and absorbance at the excitation wavelength of the standard molecule (NATEE) respectively, and Q,, F,, and A, are the analogous quantities for the analyte species. Since we are only interested in comparing the relative quantum yields of tyrosine and tryptophan fluorescence in this study, we have arbitrarily set a’. equal to unity. Absorption spectra were obtained with a computer-interfaced Cary 14 spectrophotometer (On-Line Instruments Systems Inc.). Solutions were contained in l-cm pathlength quartz cuvettes. The reported spectra are the averages of five scans each. A baseline spectrum of the buffer has been subtracted from all of the reported spectra. High minus low pH difference spectra were recorded as follows. A pair of matched l-cm quartz cuvettes were filled with 1.0 ml of 10 mM Tris buffer, pH 8.4, and used to collect a baseline absorption spectrum from 350 to 250 nm. The sample cuvette was removed and, after cleaning and drying, was filled with 1.0 ml of a ca. 2 pM solution of arrestin (in the same buffer); the absorption spectrum of this sample was recorded and stored digitally. Fifty microliters of 10 N NaOH was then added to the sample cuvette. This solution was mixed well and its absorption spectrum was recorded as above. The difference spectrum was then generated by digitally subtracting the first spectrum from the second after correcting the second spectrum for dilution by the added NaOH. Far uv circular dichroic spectra were recorded with a Jasco J-600 spectrometer which was purged with dry nitrogen gas for several hours before acquisition of data. For CD measurements the protein was dialyzed into 10 mM Tes buffer, pH 6.4. The final protein solution (ca. 200 pg/ ml arrestin) was contained in a 0.1~cm pathlength cell. Spectra were recorded from 250 to 195 nm with a scan rate of 0.2 rim/s. and a spectral bandpass of 1 nm. Arrestin was labeled with “‘1 using a modification of previously published chloramine-T-based methods (23). The reaction was performed in 10 mM sodium phosphate buffer at pH 7.5. Twenty-five microcuries of carrier-free Nan51 (ca. 0.01 nmol in 25 al of buffer), 0.1 nmol of KI (in 1 al of buffer), and 2 nmol chloramine-T (in 2 ~1 of buffer) were added sequentially to 2 nmol of arrestin dissolved in 1 ml of 10 mM Tris, pH 8.4. The reaction mixture was allowed to stand at ambient temperature for 20 min, after which the reaction was quenched by the addition of N-acetyl-L-tyrosine (1 pmol in 5 ~1 of buffer). Insulin (100 c(g) was then added to the arrestin solution as a carrier, and the labeled arrestin was isolated from free iodine, tyrosine, and insulin by size exclusion chromatography by passage through a lo-ml Bio-Bad 1ODG column. The relative binding affinity of arrestin for light-adapted rod outer segments was assessed as follows. A 0.5-ml sample of rod outer segments (76.3 FM in rhodopsin) in 70 mM sodium phosphate, 2 mM MgCl,, 0.1 mM EDTA, 3 mM ATP, 1 mM GTP, pH 7.2, was illuminated with white light from a 150-W incandescent light bulb for 10 min at 30°C. A ca. 2 pM arrestin solution containing ‘251-labeled arrestin was incubated for 5 min at the indicated temperature, and then a 0.5-ml aliquot was removed, cooled to 25”C, and added to the light-adapted rod outer segments. The resulting solution was mixed well and allowed to incubate at 3O”C, under constant illumination, for an additional 5 min. The solution was then loaded into the top of a Costar Spin-X centrifuge filter unit containing a 0.22-am cellulose acetate filter. The unit was centrifuged for 2.5 min at 13,000 rpm in a HBI microcentrifuge. After centrifugation the rod outer segments, retained on the filter, were washed with 1 ml of 10 mM Tris buffer, pH 8.4; the wash buffer was removed, through the filter, by vacuum filtration. The dried filters were placed in 16 X loo-mm glass test tubes and the radioactivity was measured with a Packard Auto-Gamma Scintillation Spectrometer for 60 s.
ET
AL.
RESULTS
As first shown by Wilden et al. (4) arrestin can be purified from retinal extract by means of its light-dependent binding to rhodopsin within rod outer segments. Isolation of the protein in this fashion results in a preparation which is approximately 95% arrestin as judged by the intensity of staining on SDS gels (Fig. 2A, lane 5). The 280-nm excited fluorescence spectrum of such a sample
10
20 Fraction
30
Number
C
J 350 Wavelength
400 (nm)
FIG. 2. (A) SDS-PAGE of bovine retinal arrestin, stained with Coomassie brilliant blue G-perchloric acid. Lane 1, fraction 18 from the final FLPC purification on a Mono-Q column. Lane 2, fraction 19 from the same column. Lane 3, fraction 20 from the same column. Lane 4, fraction 21 from the same column. Lane 5, arrestin before application to the Mono-Q column. Lane 6, molecular weight markers (Amersham Rainbow Protein Molecular Weight Markers) containing: myosin (200.0 kDa); phosphorylase b (92.5 kDa); bovine serum albumin (69.0 kDa); ovalbumin (46 kDa); carbonic anhydrase (30 kDa); trypsin inhibitor (21.5 kDa); and lysozyme (14.3 kDa). (B) Elution profile of proteins from the final Mono-Q FPLC column illustrating the 260-nm absorbance as a function of fraction number (l-ml fractions). Column conditions were as described in (16). (C) Fluorescence spectra (280 nm excitation) of arrestin fractions from the final Mono-Q FPLC column. The numbers identifying each spectrum correspond to the fraction numbers from the Mono-Q column.
CHARACTERIZATION
is characterized by a broad emission band centered at ca. 330 nm as is typical of tryptophan containing proteins (data not shown). Arrestin can be further purified by application to a Mono-Q column and elution with a O-O.5 M linear NaCl gradient (4). Figure 2B shows a typical elution pattern for the proteins from the Mono-Q column. The large Az8,, peak at fraction 20 displays a single band on SDS gels which migrates with an apparent molecular weight of 48 kDa (Fig. 2A, lane 3), as expected for bovine arrestin. In separate experiments, the main protein fractions from the Mono-Q column were subjected to SDSPAGE with silver staining; no additional protein bands were visualized in this way, supporting the inference that these fractions contain pure arrestin. As seen in Fig. 2A, fractions 18, 19, and 21 also appear to contain pure arrestin, although in lower concentration than fraction 20. The fluorescence spectra of these fractions are shown in Fig. 2C. Fraction 20, which contains the majority of the purified arrestin, gives rise to a fluorescence spectrum which is dominated by a narrow emission band centered at 303 nm. This spectrum is strikingly similar to that of N-acetyl-L-tyrosine obtained under similar conditions (data not shown) and displays little, if any, contribution from tryptophan emission. The fluorescence spectrum of fraction 21 is similar to that of fraction 20 except that the signal is diminished, as expected for the lower protein concentration in this fraction (compare lanes 3 and 4 of Fig. 2A). In contrast, the fluorescence spectra of fractions 18 and 19 are not tyrosine-like, but instead are characterized by broad emission bands centered at ca. 325 nm. Despite the difference in fluorescence features, fractions 18-21 all appear to contain pure arrestin as judged by SDS-PAGE (Fig. 2A, lanes l-4). The altered fluorescence spectra for fractions 18 and 19 may be the result of minor contaminating proteins which are not observed on SDS gels with our staining methods, or may indicate that these fractions contain denatured forms of arrestin (see below). Figure 3A illustrates the uv absorption spectrum obtained for purified arrestin (fraction 20), which exhibits the 279-nm absorption maximum typical of proteins containing both tyrosine and tryptophan. Wacker et al. (24) previously reported the extinction coefficient for this band to be 46.7 mM-’ cm-‘. In order to accurately determine the extinction coefficient for native arrestin we have made use of the tyrosinate-tyrosine difference spectrum method (25). Upon alkalinization of a tyrosine containing protein, the lowest energy K-T* transition of tyrosine is shifted from 275 to 295 nm with an accompanying increase in oscillator strength for the transition. The difference spectrum of the alkaline protein minus that at neutral pH displays a shallow trough at 275 nm and a large positive peak at ca. 295 nm, as illustrated for arrestin in Fig. 3B. The difference extinction coefficient (per tyrosine residue) for this 295-nm band varies very little among tyrosine model compounds and most proteins. Knowing the difference extinction coefficient (2.330 mM-’ cm-’ per ty-
OF
BOVINE
129
ARRESTIN
I
279
1
I
I
nn
I
(A)
:-=\:: 250
275
300
LI
,
I
I
250
275
300
325
Wavelength
FIG. 3. arrestin sorption arrestin features text for
325
350
1I 350
(nm)
(A) Ultraviolet absorption spectrum of purified bovine retinal (ca. 2 pM) in 10 mM Tris buffer, pH 8.4. (B) Ultraviolet abdifference spectrum of alkaline bovine arrestin minus bovine at pH 8.4, showing the details of the tyrosinate minus tyrosine of the spectrum. See the Materials and Methods section of the further details.
rosine residue; (25)) and the number of tyrosine residues in the amino acid sequence of arrestin, 14, (13), one can calculate the concentration of arrestin in any sample from Eq. 131 [Arrestin],
mM
=
AA2s5/(14 X 2.330),
[31
where [Arrestin], mM is the millimolar concentration of arrestin in solution, and AA2s5 is the absorbance value for the 295-nm band in the difference spectrum. Once the arrestin concentration has been thus determined one can calculate the extinction coefficient for the 279-nm band of the neutral pH spectrum by application of the BeerLambert equation (26). Evaluation of the spectra in Fig. 3 as described here results in an estimate of the extinction coefficient for bovine arrestin of 38.64 mM-’ cm-’ at 279 nm in 10 mM Tris buffer, pH 8.4. The extinction coefficient determined here is ca. 17% lower than that determined by Wacker et al. (24). We believe that the extinction coefficient determined here is more reliable since it is determined directly through spectroscopic features of the protein solutions and does not require independent determination of protein mass or lyophilization and redissolution of arrestin; we note also that estimates of protein concentration based on the present extinction coefficient were in excellent agreement with estimates for the same solutions based on the calorimetric assay of Bradford (19). Figure 4 illustrates the fluorescence spectra of bovine arrestin obtained under varying solution conditions. Fig-
KOTAKE
1.5
yArJ
0 0 0
0.1
0.2
0.3
0.4
0.
WI
300
350 Wavelength
4cQ
AL.
for collisional quenching of the 303-nm fluorescence by potassium iodide. The Stern-Volmer constant obtained from the slope of the least squares best fit of the data to a linear function is 1.19 + 0.14 M-‘, which is significantly lower than that for N-acetyl-L-tyrosine in solution (13.34 + 0.71 M-‘) but is within the range typically observed for tyrosine residues on the surface of other proteins (R. A. Copeland, unpublished data), suggesting that at least some of the tyrosine residues are solvent accessible in native arrestin. Figure 4B illustrates the 280-nm-excited fluorescence spectrum obtained for arrestin in 4 M guanidine hydrochloride (Gdn-HCl). Under these conditions two emission maxima were observed, one at ca. 303 nm and the other at ca. 340 nm. The latter emission maximum most likely represents fluorescence from tryptophan in a solvent accessible environment. When this sample was excited at 295 nm, only the 340-nm emission band was observed, confirming the assignment of this feature to tryptophan fluorescence. The changes in fluorescence brought about by Gdn-HCl are completely reversible when the Gdn-HCl concentration is diluted below ca. 0.5 M (not shown). If one computes the difference between the spectrum shown in Fig. 3B and that in Fig. 3A, one finds that the largest changes occur at 303 (negative band) and 350 nm (positive band). If one assumes that the Gdn-HCl-induced denaturation of arrestin is well modeled by a two-state transition, then the mole fraction of denatured protein can be estimated from the intensity ratio at 350/303 nm (R) as follows:
0
Lx. 1.4
LO 1.3 1.2 1.1 1.0
ET
I
(nm)
FIG. 4. Fluorescence spectra (280 nm excitation) of bovine retinal arrestin (ca. 1.5 pM) under varying solution conditions. (A) Arrestin in 10 mM Tris buffer, pH 8.4, at 25’C. Inset, Stern-Volmer plot for fluorescence quenching of the 303-nm band of arrestin by potassium iodide. (B) Arrestin in 4 M Gdn-HCl. Inset, plot of the fractional denaturation (fd) of arrestin as a function of Gdn-HCl concentration. (C) Arrestin in 10 mM Tris buffer, pH 8.4, after incubation at 79°C for 5 min and cooling to 25°C.
ure 4A shows the 280-nm excited spectrum of arrestin in 10 mM Tris buffer, pH 8.4, at 25°C. This spectrum shows tyrosine-like emission with a maximum at 303 nm. When this sample was excited at 295 nm, where one selectively excites tryptophan residues, we did not observe any significant emission above the baseline (data not shown); these data suggest that the fluorescence from the single tryptophan residue is highly quenched under these conditions.3 The inset of Fig. 4A shows a Stern-Volmer plot a In a small number of purified arrestin batches, a very weak fluorescence was observed with 295.nm excitation, with maximum emission at 339 nm. We attribute this weak emission to a minor population of denatured arrestin in these batches.
fd
=
(R
-
%)/@d
-
%I,
141
where fd is the mole fraction of denatured protein, R, is the 350/303-nm intensity ratio for the native protein (in the absence of Gdn-HCl), and Rd is the 350/303 nm intensity ratio for the fully denatured protein (estimated from the spectrum of arrestin in 6 M Gdn-HCl). The inset of Fig. 4B shows the dependence of the mole fraction of denatured arrestin on Gdn-HCl concentration. The transition midpoint estimated from this plot occurs at 1.1 M Gdn-HCl. The data from the transition region of this plot were used to calculate apparent AG values for the denaturation of arrestin at several Gdn-HCl concentrations using the relationship
AG = -RT ln[fd/(l - fd)]
[51
where R is the ideal gas constant, T is the sample temperature in degress Kelvin, and 1 - fd is the mole fraction of native protein assuming a simple two-state transition from native to denatured protein. Extrapolation of the linear plot of AG (apparent) vs. Gdn-HCl concentration to zero Gdn-HCl yields an estimate of the free energy of denaturation for arrestin, in the absence of Gdn-HCl, of +3.3 kcal/mole.
CHARACTERIZATION TABLE
OF
BOVINE
131
ARRESTIN
I
Relative Fluorescence Quantum Yields for Bovine Retinal Arrestin under Varying Solution Conditions Excitation Conditions 10 mM Tris*, 25°C 10 mM Tris, 25’C, after 5 min at 79°C 6 M Gdn-HCl 6 M Gdn-HCl 6 M Gdn-HCl
wavelength b-4
Emission wavelength (nm)
Relative
quantum yield”
280
303
1.49
280 280 280 295
325 303 340 340
0.95 0.87 0.80 1.18
20
a The relative quantum yields are referenced to the 354-nm fluorescence hand of N-acetyl-L-tryptophan ethyl ester in 10 mM Tris buffer, pH 8.0, excited at 280 nm. * The pH of all samples was maintained at 8.4.
When arrestin is incubated at 70°C for 2 5 min, the fluorescence spectrum is significantly affected (Fig. 4C). Under these conditions one observes a single emission band centered at ca. 325 nm. At this temperature, the protein solution remains clear with no evidence of any protein aggregation or precipitation. Temperature-dependent far uv circular dichroism studies suggest, however, that the protein may not be monomeric at this temperature (vide infra). The changes in arrestin fluorescence which are brought about by elevated temperature are irreversible, as shown in Fig. 4C. In this figure we show the spectrum of arrestin which has been incubated at 79°C for 5 min and then cooled back down to 25’C. The relative fluorescence quantum yields of arrestin, under varying solution conditions, are compared to that of N-acetyl-Ltryptophan ethyl ester in Table 1. Figure 5 shows the effects of elevated temperature on three properties of bovine arrestin: the fluorescence intensity ratio at 340/303 nm (open circles), the negative ellipticity at 214 nm from circular dichroic measurements (closed triangles), and the binding affinity of arrestin for light-adapted rod outer segments (closed circles). For convenient comparison of these properties, we have plotted all three as the mole fractions of denatured protein (fd) as a function of temperature. The values of fd for each property were determined in a fashion analogous to that described by Eq. [4]. It is clear from Fig. 5 that all three properties are similarly correlated to changes in temperature. The data in the transition regions of these curves were linearized by plotting In [fd/(l - fd)] as a function of temperature and the midpoints for the transitions were thus determined from least squares fits of the data. The midpoint temperatures for the fluorescence, circular dichroism, and rhodopsin binding assays were 60, 57, and 60°C, respectively.
30
40
50 60 Temperature,
70 “C
80
90
FIG. 5. Fractional denaturation (fd) of bovine retinal arrestin as a function of temperature as measured by the fluorescence intensity ratio at 340/303 nm (open circles); the binding affinity for light-adapted rod outer segments measured as the residual radioactivity on rod outer segments after incubation with rz51-labeled arrestin (which had been incubated for 5 min at the indicated temperature) and filtration through 0.22-pm cellulose acetate filters (closed circles); and the negative ellipticity at 214 nm measured by circular dichroic spectroscopy (closed triangles).
The circular dichroic spectrum of native arrestin (at 25°C) displays a broad minimum between 235 and 205 nm which, as first described by Shinohara et al. (13), reflects a significant proportion of P-sheet structure for the protein (Fig. 6). As the protein unfolds one expects the ellipticity at 214 nm to become more positive, approaching zero as the P-sheet structure is converted to random coil. When arrestin is incubated at increasingly higher temperatures, however, an enhancement of the negative ellipticity at 214 nm is observed, rather than a diminution of signal. Figure 6 illustrates this trend for the circular dichroic spectra of arrestin at four representative temperatures. These data suggest an increase in P-sheet structure for arrestin at elevated temperature. DISCUSSION The fluorescence spectrum of native arrestin is unusual for a tryptophan-containing protein in that it is dominated
buffer
-15 200
I
1
I 250
Wavelength
(nm)
FIG. 6. Temperature dependence of the far uv circular dichroic spectrum of bovine retinal arrestin. Spectra were obtained at 5°C increments from 25 to 80°C. Representative spectra of the protein at four temperatures, as indicated in the figure, are shown along with the buffer baseline obtained at 25°C.
132
KOTAKE
by tyrosine fluorescence. Usually little, if any, tyrosine emission is observed in tryptophan-containing proteins due to a variety of quenching mechanisms, including tyrosine to tryptophan resonance energy transfer (27). The fluorescence spectra reported here suggest that these quenching mechanisms are not effective in arrestin, and further suggest that the tryptophan fluorescence of this protein is highly quenched in the native protein. The mechanism of tryptophan fluorescence quenching in the native protein is not clear at this time. The extent of quenching is dependent on the protein adopting a particular folded conformation, since this quenching is relieved upon thermal or chemical denaturation of the native structure. One structural element of proteins which is thought to be an effective quencher of tryptophan fluorescence is the disulfide bond (28). Native arrestin contains one disulfide bond (3). However, treatment of arrestin with excess dithiothreitol to reduce the disulfide fails to enhance the relative quantum yield for the tryptophan residue (data not shown), suggesting that the disulfide bond is not the main cause of tryptophan quenching in this protein. Observation of a tyrosine-like fluorescence spectrum appears to be a good indicator of the purity and structural integrity of arrestin. The present data suggest that minor contaminating proteins or disruption of the native conformation of arrestin are enough to significantly alter its fluorescence spectrum, apparently by relieving the quenching of the tryptophan fluorescence. The thermal denaturation studies presented here also suggest that the tyrosine-like fluorescence spectrum is associated with the physiologically relevant conformation of arrestin, i.e., that conformation which is capable of binding to light-adapted phosphorylated rhodopsin. Fluorescence spectroscopy should therefore provide a convenient means of assessing the structural integrity of arrestin preparations from eukaryotic sources and for recombinant protein expressed in prokaryotic hosts. The temperature dependence of the circular dichroic spectrum of arrestin shows increased negative ellipticity with elevated temperature. It is well known, from a variety of model compound and protein studies, that negative ellipticity at this wavelength is indicative of P-sheet secondary structure (see for example Ref. (26)). These data therefore suggest that elevated temperature increases the P-sheet content of the protein. This was an unexpected result in that we had assumed that thermal denaturation would lead to a conformation of the protein which lacks significant secondary structure. Wacker et al. (24) have shown that arrestin self-associates into dimers at concentrations above ca.1 mg/ml. All of the experiments reported here have been conducted at a protein concentration where the vast majority of the arrestin should be present as a monomer (i.e., ~0.2 mg/ml). It is possible, however, that thermal denaturation shifts the monomerdimer equilibrium constant in favor of dimer formation.
ET AL.
As the protein unfolds at elevated temperature, hydrophobic regions of the polypeptide would become exposed to the aqueous buffer medium. The thermodynamic cost of such exposure is likely to be sufficient to drive the formation of intermolecular P-sheets between these hydrophobic regions so that they would once again be occluded from the solvent. A reasonable interpretation of the present circular dichroism data would thus be that at elevated temperature the protein unfolds and spontaneously aggregates by formation of such intermolecular P-sheet structures. This interpretation is consistent with the results of FTIR studies of the uveopathogenic peptide M derived from retinal arrestin (14), which showed that this octadecapeptide has a strong tendency to form aggregates through intermolecular ,&sheet formation. To date only three other tryptophan containing proteins have been reported in which tyrosine emission dominates the fluorescence spectrum. These are: acidic fibroblast growth factor (aFGF; Copeland et al., manuscript in preparation), basic fibroblast growth factor (bFGF, (29)), and subtilisin Carlsberg (30). Comparison of the amino acid sequences of these three proteins and bovine arrestin did not yield any common sequence that might account for their unusual fluorescence features. The only commonalities among these proteins are that they are all single tryptophan containing proteins, and all contain greater that 50% P-sheet secondary structure (Copeland et al. manuscript in preparation, (3, 31)). Recent studies suggest, however, that the presence of P-sheet structure is not sufficient to account for the tryptophan fluorescence quenching seen in these proteins (F. G. Prendergast, personal communication). Likewise, the temperature-dependent circular dichroism and fluorescence data presented here argue against P-sheet structure as the cause of fluorescence quenching in arrestin. Further analysis will be needed to determine the structural basis for the tryptophan fluorescence quenching in native arrestin. The recent cloning and expression of this protein provides a means of addressing this issue in a systematic fashion through site-directed mutagenesis (32). However, even in the absence of a detailed explanation of the quenching mechanism, it is clear that the tyrosine and tryptophan fluorescence characteristics of this protein are highly conformation dependent and suggest their usefulness as sensitive probes for arrestin conformation in solution. ACKNOWLEDGMENTS We thank Drs. M. Graziano and M. Garavito for assistance in purifying arrestin by FPLC, Drs. K. J. Willis, A. G. Szabo, M. Tota, and F. G. Prendergast for helpful discussions, and Ms. N. Ishibe for help in collecting the circular dichroic spectra. We also thank Dr. C. D. Strader for helpful discussions and for critically reading this manuscript.
REFERENCES 1. Findlay, 642.
J. B. C., and Pappin,
D. J. C. (1986)
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CHARACTERIZATION
2. Kuhn,
H., Cook, 24952502.
J. H., and Dreyer,
W. J. (1973)
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Biochemistry
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3. Kuhn, H., Hall, S. W., and Wilden, U. (1984) FE&S Lett. 176,473478. 4. Wilden, U., Hall, S. W., and Kuhn, H. (1986) Proc. N&l. Acad. Sci. USA
83,1174-1178.
5. Zuckerman, R.. and Cheasty, J. E. (1988) FEBS Lett. 238, 379384. 6. Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G., and Lefkowitz, 7.
R. J. (1990) Science 248, 1547-1550. Sibley, D. R., Benovic, J. L., Caron, (1987) Cell 48,913-922.
M.
G., and Lefkowitz,
R. J.
8. Benovic,
J. L., Kiihn, H., Weyland, I., Codina, J., Caron, M. G., and Lefkowitz, R. J. (1987) Proc. Natl. Acad. Sci. USA 84, 8879-8882. Faure, J. P. (1980) Curr. Top. Eye Res. 2, 215-302.
9. 10. Mochizuki,
M., Kuwahara, T., McAllister, C., Nussenhlatt, R. B., and Gery, I. (1985) Inuest. Ophthcdmol. Visual Sci. 17, 7744783. M., Matsumoto, H., Mirzadega, T., Ripka, 11. Asato, A. E., Denny, W. C., Crescitelli, F., and Liu, R. S. H. (1986) Biochemistry 25, 7021-7026. 12. Phillips, W. J., and Cerione, R. A. (1988) 15,505. 13. Shinohara, T., Dietzschold, B., Craft, J. J., Donoso, L. A., Horwitz, J., and Acad. Sci. USA 84,6975-6979. 14. Muga, A., Surewicz, W. K., Wong, P. (1990) Biochemistry 29, 2925-2930. 15. Teale, F. W. J. (1960) Biochem. J. 76, 16. Wilden, U., Wiirst, E., Weyand, I., and
207,292-295.
J. Biol. Chem. C. M., Wistow, Tao, R. (1987)
263,15,498G., Early, Proc. N&l.
T. T., and Mantsch, 381-388. Kuhn, H. (1986)
FEBS
H. H.
Lett.
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17. Hong,
K., and Hubbell,
W. L. (1973)
18. Laemmli,
U. K. (1970)
19. Bradford,
M. (1976)
20. Lakowicz, Plenum
Nature
Anal.
Biochem.
J. R. (1983) Principles Press, New York.
12,4517-4523.
Biochemistry
(London)
227,
680-685.
72,248-254. of Fluorescence
Spectroscopy,
21. Creighton,
T. E. (1984) Proteins: Structures and Molecular Principles, pp. 17, W. H. Freeman, New York. Parker, C. A., and Rees, W. T. (1960) The Analyst 85,587-600.
22. 23. Freychet,
P., Roth, J., and Neville, Res. Commun. 43,400-408.
J. M. (1971)
Biochem.
Biophys.
24. Wacker,
W. B., Donoso, L. A., Kalsow, C. M., Yankeelov, J. A., Jr., and Organisciak, D. T. (1977) J. Immunol. 119,1949-1958.
25. Wetlaufer, D. B. (1962) Adu. Protein Chem. 17, 303-390. 26. Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, Part
II, W. H. Freeman,
San Francisco.
27. Ross, J. B. A., Laws, H. R. (1991) Applications,
28. Cowgill,
W. R., Rousslang, K. W., and Wysslrod, in Fluorescence Spectroscopy, Volume 3: Biochemical (Lakowicz, J. R., Ed.), Plenum, New York, in press.
R. W. (1976) in Biochemical (Chen, R. F., and Edelhoch, H., Eds.), New York.
29. Arakawa, and Fox, 341.
Fluorescence Concepts 2, pp. 441-486, Marcel Dekker,
T., Hsu, Y. R., Schiffer, S. G., Tsai, L. B., Curless, C., G. M. (1989) Biochem. Biophys. Res. Commun. 161,335-
30. Willis, K. J., and Szabo, A. G. (1989) Biochemistry 28,4902-4908. 31. Bode, W., Papamokos, E., and Musil, D. (1987) Eur. J. Biochem. 166,673-692. 32. Yamaki, K., Takahashi, Y., Sakuragi, S., and Matsubara, K. (1987) Biochem. Biophys. Res. Commun. 142,904-910.
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
Physicochemical Retinal Arrestin Susan
Kotake,*
BIOPHYSICS
15, pp. 126-133,199l
Patricia
Characterization Hey,?
Raghavendra
of Bovine
G. Mirmira,*
and Robert
A. Copeland**l
*The Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, Illinois 60637; and TDepartment of Biochemistry and Molecular Pharmacology, Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
Received
September
19, 1990, and in revised
form
October
29, 1990
intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed. o ISBI Academic press, L.
The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-‘, cm-’ using the tyrosinatetyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford calorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60°C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative
The visual transduction cascade serves as a structural and mechanistic paradigm for a large family of G-proteinmediated signal transducing systems (1). In this system, light absorption by the integral membrane protein rhodopsin (Rho)’ induces a conformational transition of Rho which results in its interacting with the heterotrimeric guanine nucleotide-binding regulatory protein, transdutin. The subunits of transducin then dissociate, leading to stimulation of the activity of the retinal cGMP phosphodiesterase (1). Light absorption by Rho also triggers its phosphorylation by a specific kinase, referred to as rhodopsin kinase (2). Recently another protein, known as arrestin, S-antigen, or 48-kDa protein, has been implicated in the negative control of visual transduction (3). This protein binds to phosphorylated Rho and interferes with the interaction of Rho with transducin (4). In this way the binding of arrestin to Rho prevents the transducin-mediated stimulation of the phosphodiesterase, and the system becomes desensitized. Cross-linking studies indicate that arrestin binds not only to Rho, but also to the retinal cGMP phosphodiesterase in the presence of ATP and light (5). Thus, arrestin-mediated densensitization may be the combined
’ To whom correspondence should be addressed at Department of Biochemistry & Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, Illinois 60637.
’ Abbreviations used: Rho, rhodopsin; NATEE, IV-acetyl-L-tryptophan ethyl ester; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide electrophoresis; Gdn-HCl, guanidinium hydrochloride.
126
0003-9%x/91
Copyright All rights
0 1991 by Academic
of reproduction
in any
form
gel $3.00
Press, Inc. reserved.
CHARACTERIZATION
result of interactions of the protein with the phosphodie&erase and Rho. Desensitization of agonist responsiveness is a common feature of G-protein-coupled receptor systems. Recently, Lefkowitz and co-workers reported the cloning and expression of an arrestin-like molecule from bovine brain which appears to interact with the P-adrenergic receptor system. This protein, termed @-arrestin, binds to agonistbound phosphorylated ,8-adrenergic receptor and attenuates the interaction of the P-adrenergic receptor with the G-protein G, (6). It thus appears likely that arrestinlike molecules play a major role in the desensitization of G-protein-coupled systems in general (7, 8). In addition to its role in signal transduction, arrestin also induces experimental autoimmune uveitis (9), a T-cell-mediated disease characterized by inflammation of the retina, uveal tract, and pineal gland (10). In an effort to understand the structural basis for signal transduction in G-protein-coupled systems, biophysical methods have been used to investigate conformational transitions of the proteins in these systems, and the interactions of these proteins with one another. The conformational changes accompanying the retinal-opsin interaction have been well characterized (11). More recently, rhodopsin-transducin interactions have been analyzed by fluorescence spectroscopy (12). The solution properties of retinal arrestin are of current interest not only because of the role of this protein in signal transduction, but also because of its high pathogenicity. The secondary structure of the bovine protein has been measured by circular dichroism and analyzed by computational methods (13). In addition, Fourier transform infrared spectroscopy has shown that the uveopathogenic octadecapeptide M from retinal arrestin forms macromolecular assemblies through intermolecular P-sheet formation under physiological conditions (14). In the present study, we report the characterization of bovine retinal arrestin by a number of spectroscopic methods. The complete amino acid sequence for this protein has recently been reported (13), and is reproduced in Fig. 1. Bovine arrestin contains 14 tyrosine residues and a single tryptophan residue within its primary structure (13). We therefore expected that the fluorescence spectrum of the protein would be dominated by tryptophan emission (15). Surprisingly, however, we find that the fluorescence of the single tryptophan is quenched in the native protein, resulting in a fluorescence spectrum which is dominated by tryosine emission. The observation of this tyrosine-like fluorescence spectrum appears to be correlated with the ability of arrestin to bind to lightadapted rod outer segments. Denaturation of arrestin by either thermal or chemical means significantly perturbs the fluorescence spectrum of the protein. These unusual fluorescence properties of bovine retinal arrestin may thus prove useful in following conformational transitions of this protein in solution.
OF
BOVINE
127
ARRESTIN
MKANKPAPNH
VIFKKISRDK
LVKGKRWVS
LTCAFRYGQE
TRLQESLIKK
SVTIYLGKRD DIDVMGLSFR
VIDHVERVEP
VDGVVLVDPE
RDLYFSQVQV
FPPVGASGAT
LGANTYPFLL
TFPDYLPCSV
MLQPAWDVG
AFATHSTDVE
EDKIPKKSSV
RLLIRKVQHA
PRDMGWPRA
KPLRlAVSLS
KEIWHGEPI
PVTVAVTNST
EKTVKKIKVL
VEQVTNVVLY
SSDWIKTVA
AEEAQEKVPP
NSSLTKTLTL
VPLLANNRER
RGIALDGKIK
HEDTNLASST
IIKEGIDKTV
VPFRLh4HPCP
EDPDTAKESF
MGILVSYQIK QDENFVFEEF
VKLTVSGLLG ARQNLKDAGE
KSCGVDFEIK EASVIMFFMSD
ELTSSEVATE YKEEKlDQEA
AMDE
FIG. 1. The complete amino acid sequence for bovine retinal arrestin highlighting the aromatic amino acids tyrosine (Y) and tryptophan (W). The sequence was taken from (3).
MATERIALS
AND
METHODS
N-Acetyl-L-tryptophan ethyl ester and N-acetyl-L-tyrosine were purchased from Sigma and used as received. All other reagents were the highest quality commercially available. Dark-adapted bovine retinas were purchased from George Hormel Co. (Austin, Minnesota) and stored, in the dark, at -80°C. until use. Arrestin was purified from the dark-adapted retinas as previously described (16). Rod outer segments were purified under dim red light conditions from dark-adapted retinas, as described by Hong and Hubbell (17). Under these conditions the resulting membranes are essentially devoid of endogenous arrestin, but retain rhodopsin and rhodopsin kinase activities (16). The rhodopsin concentration of the rod outer segments was determined spectrophotometrically at 498 nm using an extinction coefficient of 42.7 mM-‘, cm-’ (17). SDS-PAGE was performed on 16% acrylamide gels by the method of Laemmli (18). Protein bands on the gels were visualized either by Coomassie brilliant blue G-perchloric acid solution or silver staining. Protein concentration was determined calorimetrically by the method of Bradford (19) using either y-globulin or bovine serum albumin to obtain a standard curve. Fluorescence spectra were obtained with either a PTI alphascan or SLM 48000s spectrofluorometer. The samples were contained in a lcm quartz cuvette which was placed in a temperature-controlled sample holder. The temperature of the sample was maintained at 25’C throughout the experiments by circulating a water/ethylene glycol mixture through the sample holder from a refrigerated water bath. All of the reported spectra were obtained with the emission polarizer set at 54.75” to minimize spectral anomalies due to polarization bias and stray light scattering (20). Emission intensities were corrected for variation in instrument response by reference to a 3 g/liter solution of rhodamine B in ethylene glycol (20). The reported spectra have not been corrected for inner filter effects, but in all cases the absorptivity of the sample was kept below 0.1 to minimize such effects. The relative quantum yields of arrestin tyrosine and tryptophan fluorescence, reported in Table I, are referenced to the 354-nm emission band of a 5 pM solution of N-acetyl-L-tryptophan ethyl ester (NATEE) in 10 mM Tris-HCI buffer, pH 8.0, excited with 280-nm light. The NATEE solution was prepared by dilution of a 100 pM stock solution whose concentration was determined spectrophotometrically using an extinction coefficient of 5,600 M-km-’ at 280 nm (21). The quantum yield, 9, of a fluorophore can be related to the observed fluorescence intensity as follows (22):
F = Z,,(2.3 cd) a, where F is the fluorescence intensity, Z0 is the incident light intensity, e is the molar extinction coefficient for the fluorophore at the excitation
111
128
KOTAKE
wavelength, c is the sample concentration, and d is the pathlength of the cuvette. Note that the product ccd is equivalent to the absorbance of the sample at the excitation wavelength. If the quantities I0 and d are held constant, the quantum yield of any analyte can be determined by measuring its fluorescence spectrum along with the spectrum of a standard of known quantum yield, and using equation (22)
where a,, F,, and A, are the quantum yield, fluorescence intensity, and absorbance at the excitation wavelength of the standard molecule (NATEE) respectively, and Q,, F,, and A, are the analogous quantities for the analyte species. Since we are only interested in comparing the relative quantum yields of tyrosine and tryptophan fluorescence in this study, we have arbitrarily set a’. equal to unity. Absorption spectra were obtained with a computer-interfaced Cary 14 spectrophotometer (On-Line Instruments Systems Inc.). Solutions were contained in l-cm pathlength quartz cuvettes. The reported spectra are the averages of five scans each. A baseline spectrum of the buffer has been subtracted from all of the reported spectra. High minus low pH difference spectra were recorded as follows. A pair of matched l-cm quartz cuvettes were filled with 1.0 ml of 10 mM Tris buffer, pH 8.4, and used to collect a baseline absorption spectrum from 350 to 250 nm. The sample cuvette was removed and, after cleaning and drying, was filled with 1.0 ml of a ca. 2 pM solution of arrestin (in the same buffer); the absorption spectrum of this sample was recorded and stored digitally. Fifty microliters of 10 N NaOH was then added to the sample cuvette. This solution was mixed well and its absorption spectrum was recorded as above. The difference spectrum was then generated by digitally subtracting the first spectrum from the second after correcting the second spectrum for dilution by the added NaOH. Far uv circular dichroic spectra were recorded with a Jasco J-600 spectrometer which was purged with dry nitrogen gas for several hours before acquisition of data. For CD measurements the protein was dialyzed into 10 mM Tes buffer, pH 6.4. The final protein solution (ca. 200 pg/ ml arrestin) was contained in a 0.1~cm pathlength cell. Spectra were recorded from 250 to 195 nm with a scan rate of 0.2 rim/s. and a spectral bandpass of 1 nm. Arrestin was labeled with “‘1 using a modification of previously published chloramine-T-based methods (23). The reaction was performed in 10 mM sodium phosphate buffer at pH 7.5. Twenty-five microcuries of carrier-free Nan51 (ca. 0.01 nmol in 25 al of buffer), 0.1 nmol of KI (in 1 al of buffer), and 2 nmol chloramine-T (in 2 ~1 of buffer) were added sequentially to 2 nmol of arrestin dissolved in 1 ml of 10 mM Tris, pH 8.4. The reaction mixture was allowed to stand at ambient temperature for 20 min, after which the reaction was quenched by the addition of N-acetyl-L-tyrosine (1 pmol in 5 ~1 of buffer). Insulin (100 c(g) was then added to the arrestin solution as a carrier, and the labeled arrestin was isolated from free iodine, tyrosine, and insulin by size exclusion chromatography by passage through a lo-ml Bio-Bad 1ODG column. The relative binding affinity of arrestin for light-adapted rod outer segments was assessed as follows. A 0.5-ml sample of rod outer segments (76.3 FM in rhodopsin) in 70 mM sodium phosphate, 2 mM MgCl,, 0.1 mM EDTA, 3 mM ATP, 1 mM GTP, pH 7.2, was illuminated with white light from a 150-W incandescent light bulb for 10 min at 30°C. A ca. 2 pM arrestin solution containing ‘251-labeled arrestin was incubated for 5 min at the indicated temperature, and then a 0.5-ml aliquot was removed, cooled to 25”C, and added to the light-adapted rod outer segments. The resulting solution was mixed well and allowed to incubate at 3O”C, under constant illumination, for an additional 5 min. The solution was then loaded into the top of a Costar Spin-X centrifuge filter unit containing a 0.22-am cellulose acetate filter. The unit was centrifuged for 2.5 min at 13,000 rpm in a HBI microcentrifuge. After centrifugation the rod outer segments, retained on the filter, were washed with 1 ml of 10 mM Tris buffer, pH 8.4; the wash buffer was removed, through the filter, by vacuum filtration. The dried filters were placed in 16 X loo-mm glass test tubes and the radioactivity was measured with a Packard Auto-Gamma Scintillation Spectrometer for 60 s.
ET
AL.
RESULTS
As first shown by Wilden et al. (4) arrestin can be purified from retinal extract by means of its light-dependent binding to rhodopsin within rod outer segments. Isolation of the protein in this fashion results in a preparation which is approximately 95% arrestin as judged by the intensity of staining on SDS gels (Fig. 2A, lane 5). The 280-nm excited fluorescence spectrum of such a sample
10
20 Fraction
30
Number
C
J 350 Wavelength
400 (nm)
FIG. 2. (A) SDS-PAGE of bovine retinal arrestin, stained with Coomassie brilliant blue G-perchloric acid. Lane 1, fraction 18 from the final FLPC purification on a Mono-Q column. Lane 2, fraction 19 from the same column. Lane 3, fraction 20 from the same column. Lane 4, fraction 21 from the same column. Lane 5, arrestin before application to the Mono-Q column. Lane 6, molecular weight markers (Amersham Rainbow Protein Molecular Weight Markers) containing: myosin (200.0 kDa); phosphorylase b (92.5 kDa); bovine serum albumin (69.0 kDa); ovalbumin (46 kDa); carbonic anhydrase (30 kDa); trypsin inhibitor (21.5 kDa); and lysozyme (14.3 kDa). (B) Elution profile of proteins from the final Mono-Q FPLC column illustrating the 260-nm absorbance as a function of fraction number (l-ml fractions). Column conditions were as described in (16). (C) Fluorescence spectra (280 nm excitation) of arrestin fractions from the final Mono-Q FPLC column. The numbers identifying each spectrum correspond to the fraction numbers from the Mono-Q column.
CHARACTERIZATION
is characterized by a broad emission band centered at ca. 330 nm as is typical of tryptophan containing proteins (data not shown). Arrestin can be further purified by application to a Mono-Q column and elution with a O-O.5 M linear NaCl gradient (4). Figure 2B shows a typical elution pattern for the proteins from the Mono-Q column. The large Az8,, peak at fraction 20 displays a single band on SDS gels which migrates with an apparent molecular weight of 48 kDa (Fig. 2A, lane 3), as expected for bovine arrestin. In separate experiments, the main protein fractions from the Mono-Q column were subjected to SDSPAGE with silver staining; no additional protein bands were visualized in this way, supporting the inference that these fractions contain pure arrestin. As seen in Fig. 2A, fractions 18, 19, and 21 also appear to contain pure arrestin, although in lower concentration than fraction 20. The fluorescence spectra of these fractions are shown in Fig. 2C. Fraction 20, which contains the majority of the purified arrestin, gives rise to a fluorescence spectrum which is dominated by a narrow emission band centered at 303 nm. This spectrum is strikingly similar to that of N-acetyl-L-tyrosine obtained under similar conditions (data not shown) and displays little, if any, contribution from tryptophan emission. The fluorescence spectrum of fraction 21 is similar to that of fraction 20 except that the signal is diminished, as expected for the lower protein concentration in this fraction (compare lanes 3 and 4 of Fig. 2A). In contrast, the fluorescence spectra of fractions 18 and 19 are not tyrosine-like, but instead are characterized by broad emission bands centered at ca. 325 nm. Despite the difference in fluorescence features, fractions 18-21 all appear to contain pure arrestin as judged by SDS-PAGE (Fig. 2A, lanes l-4). The altered fluorescence spectra for fractions 18 and 19 may be the result of minor contaminating proteins which are not observed on SDS gels with our staining methods, or may indicate that these fractions contain denatured forms of arrestin (see below). Figure 3A illustrates the uv absorption spectrum obtained for purified arrestin (fraction 20), which exhibits the 279-nm absorption maximum typical of proteins containing both tyrosine and tryptophan. Wacker et al. (24) previously reported the extinction coefficient for this band to be 46.7 mM-’ cm-‘. In order to accurately determine the extinction coefficient for native arrestin we have made use of the tyrosinate-tyrosine difference spectrum method (25). Upon alkalinization of a tyrosine containing protein, the lowest energy K-T* transition of tyrosine is shifted from 275 to 295 nm with an accompanying increase in oscillator strength for the transition. The difference spectrum of the alkaline protein minus that at neutral pH displays a shallow trough at 275 nm and a large positive peak at ca. 295 nm, as illustrated for arrestin in Fig. 3B. The difference extinction coefficient (per tyrosine residue) for this 295-nm band varies very little among tyrosine model compounds and most proteins. Knowing the difference extinction coefficient (2.330 mM-’ cm-’ per ty-
OF
BOVINE
129
ARRESTIN
I
279
1
I
I
nn
I
(A)
:-=\:: 250
275
300
LI
,
I
I
250
275
300
325
Wavelength
FIG. 3. arrestin sorption arrestin features text for
325
350
1I 350
(nm)
(A) Ultraviolet absorption spectrum of purified bovine retinal (ca. 2 pM) in 10 mM Tris buffer, pH 8.4. (B) Ultraviolet abdifference spectrum of alkaline bovine arrestin minus bovine at pH 8.4, showing the details of the tyrosinate minus tyrosine of the spectrum. See the Materials and Methods section of the further details.
rosine residue; (25)) and the number of tyrosine residues in the amino acid sequence of arrestin, 14, (13), one can calculate the concentration of arrestin in any sample from Eq. 131 [Arrestin],
mM
=
AA2s5/(14 X 2.330),
[31
where [Arrestin], mM is the millimolar concentration of arrestin in solution, and AA2s5 is the absorbance value for the 295-nm band in the difference spectrum. Once the arrestin concentration has been thus determined one can calculate the extinction coefficient for the 279-nm band of the neutral pH spectrum by application of the BeerLambert equation (26). Evaluation of the spectra in Fig. 3 as described here results in an estimate of the extinction coefficient for bovine arrestin of 38.64 mM-’ cm-’ at 279 nm in 10 mM Tris buffer, pH 8.4. The extinction coefficient determined here is ca. 17% lower than that determined by Wacker et al. (24). We believe that the extinction coefficient determined here is more reliable since it is determined directly through spectroscopic features of the protein solutions and does not require independent determination of protein mass or lyophilization and redissolution of arrestin; we note also that estimates of protein concentration based on the present extinction coefficient were in excellent agreement with estimates for the same solutions based on the calorimetric assay of Bradford (19). Figure 4 illustrates the fluorescence spectra of bovine arrestin obtained under varying solution conditions. Fig-
KOTAKE
1.5
yArJ
0 0 0
0.1
0.2
0.3
0.4
0.
WI
300
350 Wavelength
4cQ
AL.
for collisional quenching of the 303-nm fluorescence by potassium iodide. The Stern-Volmer constant obtained from the slope of the least squares best fit of the data to a linear function is 1.19 + 0.14 M-‘, which is significantly lower than that for N-acetyl-L-tyrosine in solution (13.34 + 0.71 M-‘) but is within the range typically observed for tyrosine residues on the surface of other proteins (R. A. Copeland, unpublished data), suggesting that at least some of the tyrosine residues are solvent accessible in native arrestin. Figure 4B illustrates the 280-nm-excited fluorescence spectrum obtained for arrestin in 4 M guanidine hydrochloride (Gdn-HCl). Under these conditions two emission maxima were observed, one at ca. 303 nm and the other at ca. 340 nm. The latter emission maximum most likely represents fluorescence from tryptophan in a solvent accessible environment. When this sample was excited at 295 nm, only the 340-nm emission band was observed, confirming the assignment of this feature to tryptophan fluorescence. The changes in fluorescence brought about by Gdn-HCl are completely reversible when the Gdn-HCl concentration is diluted below ca. 0.5 M (not shown). If one computes the difference between the spectrum shown in Fig. 3B and that in Fig. 3A, one finds that the largest changes occur at 303 (negative band) and 350 nm (positive band). If one assumes that the Gdn-HCl-induced denaturation of arrestin is well modeled by a two-state transition, then the mole fraction of denatured protein can be estimated from the intensity ratio at 350/303 nm (R) as follows:
0
Lx. 1.4
LO 1.3 1.2 1.1 1.0
ET
I
(nm)
FIG. 4. Fluorescence spectra (280 nm excitation) of bovine retinal arrestin (ca. 1.5 pM) under varying solution conditions. (A) Arrestin in 10 mM Tris buffer, pH 8.4, at 25’C. Inset, Stern-Volmer plot for fluorescence quenching of the 303-nm band of arrestin by potassium iodide. (B) Arrestin in 4 M Gdn-HCl. Inset, plot of the fractional denaturation (fd) of arrestin as a function of Gdn-HCl concentration. (C) Arrestin in 10 mM Tris buffer, pH 8.4, after incubation at 79°C for 5 min and cooling to 25°C.
ure 4A shows the 280-nm excited spectrum of arrestin in 10 mM Tris buffer, pH 8.4, at 25°C. This spectrum shows tyrosine-like emission with a maximum at 303 nm. When this sample was excited at 295 nm, where one selectively excites tryptophan residues, we did not observe any significant emission above the baseline (data not shown); these data suggest that the fluorescence from the single tryptophan residue is highly quenched under these conditions.3 The inset of Fig. 4A shows a Stern-Volmer plot a In a small number of purified arrestin batches, a very weak fluorescence was observed with 295.nm excitation, with maximum emission at 339 nm. We attribute this weak emission to a minor population of denatured arrestin in these batches.
fd
=
(R
-
%)/@d
-
%I,
141
where fd is the mole fraction of denatured protein, R, is the 350/303-nm intensity ratio for the native protein (in the absence of Gdn-HCl), and Rd is the 350/303 nm intensity ratio for the fully denatured protein (estimated from the spectrum of arrestin in 6 M Gdn-HCl). The inset of Fig. 4B shows the dependence of the mole fraction of denatured arrestin on Gdn-HCl concentration. The transition midpoint estimated from this plot occurs at 1.1 M Gdn-HCl. The data from the transition region of this plot were used to calculate apparent AG values for the denaturation of arrestin at several Gdn-HCl concentrations using the relationship
AG = -RT ln[fd/(l - fd)]
[51
where R is the ideal gas constant, T is the sample temperature in degress Kelvin, and 1 - fd is the mole fraction of native protein assuming a simple two-state transition from native to denatured protein. Extrapolation of the linear plot of AG (apparent) vs. Gdn-HCl concentration to zero Gdn-HCl yields an estimate of the free energy of denaturation for arrestin, in the absence of Gdn-HCl, of +3.3 kcal/mole.
CHARACTERIZATION TABLE
OF
BOVINE
131
ARRESTIN
I
Relative Fluorescence Quantum Yields for Bovine Retinal Arrestin under Varying Solution Conditions Excitation Conditions 10 mM Tris*, 25°C 10 mM Tris, 25’C, after 5 min at 79°C 6 M Gdn-HCl 6 M Gdn-HCl 6 M Gdn-HCl
wavelength b-4
Emission wavelength (nm)
Relative
quantum yield”
280
303
1.49
280 280 280 295
325 303 340 340
0.95 0.87 0.80 1.18
20
a The relative quantum yields are referenced to the 354-nm fluorescence hand of N-acetyl-L-tryptophan ethyl ester in 10 mM Tris buffer, pH 8.0, excited at 280 nm. * The pH of all samples was maintained at 8.4.
When arrestin is incubated at 70°C for 2 5 min, the fluorescence spectrum is significantly affected (Fig. 4C). Under these conditions one observes a single emission band centered at ca. 325 nm. At this temperature, the protein solution remains clear with no evidence of any protein aggregation or precipitation. Temperature-dependent far uv circular dichroism studies suggest, however, that the protein may not be monomeric at this temperature (vide infra). The changes in arrestin fluorescence which are brought about by elevated temperature are irreversible, as shown in Fig. 4C. In this figure we show the spectrum of arrestin which has been incubated at 79°C for 5 min and then cooled back down to 25’C. The relative fluorescence quantum yields of arrestin, under varying solution conditions, are compared to that of N-acetyl-Ltryptophan ethyl ester in Table 1. Figure 5 shows the effects of elevated temperature on three properties of bovine arrestin: the fluorescence intensity ratio at 340/303 nm (open circles), the negative ellipticity at 214 nm from circular dichroic measurements (closed triangles), and the binding affinity of arrestin for light-adapted rod outer segments (closed circles). For convenient comparison of these properties, we have plotted all three as the mole fractions of denatured protein (fd) as a function of temperature. The values of fd for each property were determined in a fashion analogous to that described by Eq. [4]. It is clear from Fig. 5 that all three properties are similarly correlated to changes in temperature. The data in the transition regions of these curves were linearized by plotting In [fd/(l - fd)] as a function of temperature and the midpoints for the transitions were thus determined from least squares fits of the data. The midpoint temperatures for the fluorescence, circular dichroism, and rhodopsin binding assays were 60, 57, and 60°C, respectively.
30
40
50 60 Temperature,
70 “C
80
90
FIG. 5. Fractional denaturation (fd) of bovine retinal arrestin as a function of temperature as measured by the fluorescence intensity ratio at 340/303 nm (open circles); the binding affinity for light-adapted rod outer segments measured as the residual radioactivity on rod outer segments after incubation with rz51-labeled arrestin (which had been incubated for 5 min at the indicated temperature) and filtration through 0.22-pm cellulose acetate filters (closed circles); and the negative ellipticity at 214 nm measured by circular dichroic spectroscopy (closed triangles).
The circular dichroic spectrum of native arrestin (at 25°C) displays a broad minimum between 235 and 205 nm which, as first described by Shinohara et al. (13), reflects a significant proportion of P-sheet structure for the protein (Fig. 6). As the protein unfolds one expects the ellipticity at 214 nm to become more positive, approaching zero as the P-sheet structure is converted to random coil. When arrestin is incubated at increasingly higher temperatures, however, an enhancement of the negative ellipticity at 214 nm is observed, rather than a diminution of signal. Figure 6 illustrates this trend for the circular dichroic spectra of arrestin at four representative temperatures. These data suggest an increase in P-sheet structure for arrestin at elevated temperature. DISCUSSION The fluorescence spectrum of native arrestin is unusual for a tryptophan-containing protein in that it is dominated
buffer
-15 200
I
1
I 250
Wavelength
(nm)
FIG. 6. Temperature dependence of the far uv circular dichroic spectrum of bovine retinal arrestin. Spectra were obtained at 5°C increments from 25 to 80°C. Representative spectra of the protein at four temperatures, as indicated in the figure, are shown along with the buffer baseline obtained at 25°C.
132
KOTAKE
by tyrosine fluorescence. Usually little, if any, tyrosine emission is observed in tryptophan-containing proteins due to a variety of quenching mechanisms, including tyrosine to tryptophan resonance energy transfer (27). The fluorescence spectra reported here suggest that these quenching mechanisms are not effective in arrestin, and further suggest that the tryptophan fluorescence of this protein is highly quenched in the native protein. The mechanism of tryptophan fluorescence quenching in the native protein is not clear at this time. The extent of quenching is dependent on the protein adopting a particular folded conformation, since this quenching is relieved upon thermal or chemical denaturation of the native structure. One structural element of proteins which is thought to be an effective quencher of tryptophan fluorescence is the disulfide bond (28). Native arrestin contains one disulfide bond (3). However, treatment of arrestin with excess dithiothreitol to reduce the disulfide fails to enhance the relative quantum yield for the tryptophan residue (data not shown), suggesting that the disulfide bond is not the main cause of tryptophan quenching in this protein. Observation of a tyrosine-like fluorescence spectrum appears to be a good indicator of the purity and structural integrity of arrestin. The present data suggest that minor contaminating proteins or disruption of the native conformation of arrestin are enough to significantly alter its fluorescence spectrum, apparently by relieving the quenching of the tryptophan fluorescence. The thermal denaturation studies presented here also suggest that the tyrosine-like fluorescence spectrum is associated with the physiologically relevant conformation of arrestin, i.e., that conformation which is capable of binding to light-adapted phosphorylated rhodopsin. Fluorescence spectroscopy should therefore provide a convenient means of assessing the structural integrity of arrestin preparations from eukaryotic sources and for recombinant protein expressed in prokaryotic hosts. The temperature dependence of the circular dichroic spectrum of arrestin shows increased negative ellipticity with elevated temperature. It is well known, from a variety of model compound and protein studies, that negative ellipticity at this wavelength is indicative of P-sheet secondary structure (see for example Ref. (26)). These data therefore suggest that elevated temperature increases the P-sheet content of the protein. This was an unexpected result in that we had assumed that thermal denaturation would lead to a conformation of the protein which lacks significant secondary structure. Wacker et al. (24) have shown that arrestin self-associates into dimers at concentrations above ca.1 mg/ml. All of the experiments reported here have been conducted at a protein concentration where the vast majority of the arrestin should be present as a monomer (i.e., ~0.2 mg/ml). It is possible, however, that thermal denaturation shifts the monomerdimer equilibrium constant in favor of dimer formation.
ET AL.
As the protein unfolds at elevated temperature, hydrophobic regions of the polypeptide would become exposed to the aqueous buffer medium. The thermodynamic cost of such exposure is likely to be sufficient to drive the formation of intermolecular P-sheets between these hydrophobic regions so that they would once again be occluded from the solvent. A reasonable interpretation of the present circular dichroism data would thus be that at elevated temperature the protein unfolds and spontaneously aggregates by formation of such intermolecular P-sheet structures. This interpretation is consistent with the results of FTIR studies of the uveopathogenic peptide M derived from retinal arrestin (14), which showed that this octadecapeptide has a strong tendency to form aggregates through intermolecular ,&sheet formation. To date only three other tryptophan containing proteins have been reported in which tyrosine emission dominates the fluorescence spectrum. These are: acidic fibroblast growth factor (aFGF; Copeland et al., manuscript in preparation), basic fibroblast growth factor (bFGF, (29)), and subtilisin Carlsberg (30). Comparison of the amino acid sequences of these three proteins and bovine arrestin did not yield any common sequence that might account for their unusual fluorescence features. The only commonalities among these proteins are that they are all single tryptophan containing proteins, and all contain greater that 50% P-sheet secondary structure (Copeland et al. manuscript in preparation, (3, 31)). Recent studies suggest, however, that the presence of P-sheet structure is not sufficient to account for the tryptophan fluorescence quenching seen in these proteins (F. G. Prendergast, personal communication). Likewise, the temperature-dependent circular dichroism and fluorescence data presented here argue against P-sheet structure as the cause of fluorescence quenching in arrestin. Further analysis will be needed to determine the structural basis for the tryptophan fluorescence quenching in native arrestin. The recent cloning and expression of this protein provides a means of addressing this issue in a systematic fashion through site-directed mutagenesis (32). However, even in the absence of a detailed explanation of the quenching mechanism, it is clear that the tyrosine and tryptophan fluorescence characteristics of this protein are highly conformation dependent and suggest their usefulness as sensitive probes for arrestin conformation in solution. ACKNOWLEDGMENTS We thank Drs. M. Graziano and M. Garavito for assistance in purifying arrestin by FPLC, Drs. K. J. Willis, A. G. Szabo, M. Tota, and F. G. Prendergast for helpful discussions, and Ms. N. Ishibe for help in collecting the circular dichroic spectra. We also thank Dr. C. D. Strader for helpful discussions and for critically reading this manuscript.
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