Characterization of the tryptophan environments of interleukins 1α and 1β by fluorescence quenching and lifetime measurements

ARCHIVESOFBIOCHEMISTRYAND BIOPHYSICS Vol. 275, No. 1, November 15, pp. 82-91,1989

Characterization of the Tryptophan Environments of lnterleukins 1 a! and 1p by Fluorescence Quenching and Lifetime Measurements DENNIS

E. EPPS,*,l ANTHONY

W. YEM,? AND MARTIN

R. DEIBEL,

*Physical and Analytical Chemistry and TBiopolymer Chemistry, The Upjohn 7000Portage, Kalamazoo, Michigan 49001

JR.+

Company,

Received April 3,1989, and in revised form July 17,1989

The tryptophan environments of interleukins la and lp, immunomodulatory proteins with similar biological activities but only 25% sequence homology, were characterized by steady-state and dynamic fluorescence measurements. Both proteins exhibited similar emission maxima, but the emission intensity of IL-l/3 was greatly enhanced by increasing the ionic strength of the medium, whereas that of IL-la was unaffected, The two cytokines were also similarly quenched by the polar quencher acrylamide, but differences were observed for the ionic quenchers iodide and cesium. The fluorescence intensity decays of both cytokines were characterized by two (long and short) component lifetimes. However, the average lifetime of IL-l/3 (4.4 ns) was much longer than that of IL-la (1.93 ns). Taken together with the results of steady-state measurements, we suggest that the single tryptophan of IL-18 is statically quenched by neighboring charged residues, whereas the tryptophan fluorescence of IL-la is unaffected by ionic strength, and that the tryptophans of the two proteins have different accessibilities to ionic quenchers. The results are discussed in terms of similarities and differences in the tryptophan environments of the two proteins. o 1989AcademicPROS,IN.

Interleukin 1 (IL-l)’ is an immunomodulatory protein which is involved in the regulation of immunological and inflammatory processes (for review, see Ref. (1)). It is one of the key mediators of the body’s response to microbial invasion, inflammation, immunological reactions, and tissue injury. Both natural and recombinant IL1 proteins produce fever, stimulate PGEa, and induce the proliferation of fibroblasts and T lymphocytes (2). Specifically, IL-l is a member of the class of cellular mediators known as cytokines, which can be produced by virtually every nucleated cell type. Cloning of human IL-l cDNAs has resulted in the production of two human macrophage-derived sequences (3), which have been named IL-lo and IL-l@. Large quantities of pure human IL-l@ and IL-la are

now available as a result of high level expression in Escherichia coli of the respective cDNAs (4). The mature extracellular form of IL-l/3 comprises 153 amino acid residues, a molecular mass of 17,000 Da, a single tryptophan residue, and two free cysteines (5). In contrast, IL-la is slightly larger, comprising 159 amino acid residues, a molecular mass of 18,000 Da, two tryptophan residues, and a single free cysteine (6). The two proteins have only 25% sequence homology, but both proteins are reported to bind to the same receptor and produce similar biological responses (7,8). Recombinant interleukin 18 expressed in E. coli has a single tryptophan residue at position 120 and four tyrosines (positions 24,68, 90, and 121). Recombinant interleukin la, similarly expressed in E. coli has two tryptophan residues (positions 113 and 139) and seven tyrosines (12,20,39,59, 80,121, and 138). Since the minimum polypeptide length of the functionally active

’ To whom correspondence should be addressed. *Abbreviations used: IL-l, interleukin 1; SDS, sodium dodecyl sulfate. 0003-986149 $3.00

CopYright0 1989by AcademicPress,Inc. All rights of reproductionin any form reserved.

82

FLUORESCENCE

PROPERTIES OF INTERLEUKINS

sites of IL-la and IL-10 are 16-155 and 4150, respectively, the tryptophans of both proteins are in regions essential for biological activity (16). Although the sequence homology of interleukins IL-la and IL-lb is only 25%, it is likely that the three-dimensional structures of the proteins would have to be somewhat similar since they produce very similar biological effects. Recently, the crystal structure of interleukin l@ (to 3 A resolution) was reported (17). The protein was found to have a tetrahedron-like core, but no coordinates were published, nor was any information as to the structure of the tryptophan region given. The mechanism of action of IL-l remains elusive; it is known to bind to its receptor sites on a variety of cell types, but little is known concerning the specific interaction of IL-l(s) with its receptor (for review, see Ref. (9)). The specific conformations of the interleukins are responsible for their binding with high affinity to specific receptors, and hence their biological activity. A recent proton NMR study of the aromatic residues of IL-lp and mutants has been published (lo), but the authors did not present specific structural data concerning the tryptophan environment of the protein. In addition, conformation, folding, and stability characteristics of ILlp were explored by a combination of CD, uv/vis, and fluorescence spectroscopy (11). This study, however, was primarily concerned with folding characteristics of the protein, and characterization of the tryptophan environment was minimal. However, the authors were able to conclude that the single tryptophan is 40% exposed to solvent from solvent perturbation experiments. Fluorescence spectroscopy is a useful technique for quickly obtaining useful information about the conformation of proteins. Specifically, the fluorescence characteristics of intrinsic tryptophan residues of proteins can sometimes provide extensive information about the folding and solution behavior of the macromolecule. Fluorescence quenching by penetrating and ionic collisional agents can be further utilized to probe the environment around intrinsic tryptophan residues. Additional

la AND 16

83

information can be obtained from fluorescence lifetime measurements of the intrinsic tryptophan fluorescence. It is of importance to define the active site(s) of the IL-l molecule in order to understand its interaction with the IL-1 receptor and to determine if the multiple biological functions are due to multiple structural domains of the protein(s). The tryptophan(s) of IL-la and IL-l@ reside within the minimal segment of the protein required for biological activity. In the present work, we have utilized ionic and penetrating quenching agents, and dynamic measurements of the intrinsic tryptophan fluorescence to assess conformational differences and similarities between IL-la and IL-l& We find major differences in the accessibility and environments of tryptophan residues of the two proteins. The results are discussed in terms of possible overall conformations of the two interleukins. MATERIALS

AND METHODS

Chemicals. Acrylamide and pterphenyl were from Aldrich Chemical Co., and Kl, KCI, NaCI, and C&l were from Fisher Chemicals. All chemicals were reagent grade. Intedeukins. Interleukin lol (recombinant human protein expressed in E. cdi) was obtained from Dainippon (6). Its amino acid sequence had been confirmed chemically by Edman degradation and enzymatically using carboxypeptidase; N-terminal analysis showed that the amino-terminal amino acid was serine, indicating the removal of a methionine residue due to the initiation eodon. The protein has a molecular mass of 18,000Da, gives a single band by SDSpolyacrylamide gel electrophoresis, and has an isoelectric point of 5.3. It was found to have an endotoxin level of 1.63 ng/mg protein and was stored in 5 mM phosphate-buffered saline at -25°C until use. When stored in high concentration for extended periods of time at 4°C. the protein was found to dimerize, due to the formation of intermolecular disulfide bonds between the single cysteine residues (see Results). Interleukin l/3 was cloned, expressed in E. co& and purified at The Upjohn Company as recently described (4). The protein activity was stable when stored at 4°C in 10 mM Tris-HCl, pH 9.0, 10% glycerol, 0.15M NaCl. Both interleukin lcu and 10 were dialyzed before use to remove salt and/or glycerol. SDS-pol~acrylamide gel electrophoresti. Gel electrophoresis was conducted according to the procedure of Laemmli (12). Gels were polymerized to contain 18% polyacrylamide (3’7.5:l.O acrylamide:bisacryl-

84

EPPS, YEM, AND DEIBEL

amide ratio) and run in a Protean II gel system (BioRad Laboratories). Samples were diluted 1:l with sample denaturation buffer (0.25 M Tris-HCI, pH 6.8, 2% SDS, 50 mM dithiothreitol, and 25% glycerol) and immersed in a boiling water bath for 2 min. Samples were electrophoresed at a constant power of 5 W for 6 h at room temperature, and stained with Coomassie brilliant blue G-250 according to the method of Holbrook and Leaver (13). To identify dimeric and oligomerit species of the proteins mediated by disulfide bridge formation, samples were also pretreated with sample denaturation buffer (as above) but in the absence of dithiothreitol. Quantitation of dimeric species was determined by densitometric analyses of dye-stained SDS polyacrylamide gels. Fluorescent measurements: Steady state. Fluorescence measurements were made in the ratio mode on either an SLM 500C or Perkin-Elmer MPF-66 instrument, using the spectral software supplied by the manufacturers. A constant temperature of 25°C was maintained by using either a Lauda or Neslab circulating temperature bath. Emission spectra (uncorrected) were taken with either 2- or 4-nm excitation slits and 5- or 7-nm emission slits. The excitation wavelength was 295 or 275 nm for the emission scans. Emission spectra blanks (buffer only) were run and subtracted from the emission spectra of the proteins. Quenching agents were added from 2.5 M stock solutions and the contents of the cuvettes (protein = 20 pg/ml in 0.01 M phosphate, pH 7.28) mixed by inversion or by mild stirring (IL-l@ with a small stir bar. Sodium thiosulfate (0.1%) was added to stockK1 solutions to prevent the formation of 12.The fluorescence intensity was measured as a function of quencher concentration at a fixed emission wavelength of 340 nm with excitation at 295 nm. All fluorescence intensity measurements were corrected for the dilution effect resulting from addition of quencher. In the case of acrylamide quenching, an additional correction was made to account for absorbance by acrylamide at the wavelengths used. The correction employed was according to Lakowicz (14), F eOrr= FobsX antilog((&

+ &J/2),

Dl

where F,,, and Fobsare the corrected and observed fluorescence intensities at 340 nm and A,, and A,, are the absorbances of the solutions at the excitation and emission wavelengths, respectively (as measured with a Perkin-Elmer Lambda V spectrophotometer). The quenching data were analyzed by the well-known Stern Volmer equation,

FdF = 1 + KJQI,

FdAF = ~MJ,JQl) + l/f,, where f, is the fraction of the initial fluorescence which is accessible to quencher. The y intercept in this Lehrer (15) plot is the reciprocal of the percentage accessibility of the tryptophan residue to the quenching agent, and the slope is l/f KBY.Only modified Stern Volmer plots are shown in the figures. Flwrescence lifetime measurements. Multifrequency fluorescence lifetime measurements of the intrinsic tryptophan fluorescence of interleukins 10 and l@ (40 pg/ml in 0.01 phosphate, pH 7.28) were made using a cross-correlation phase and modulation fluorometer. The harmonic content of a high repetition rate mode-locked Nd-YAG laser was used to generate selected frequencies from 8 to 170 MHzl. The NdYAG laser (Spectra Physics, Inc.) was used to synchronously pump a dye laser (Rhodamine 590) whose pulse train was frequency doubled with an angle tuned frequency doubler utilizing a KDP crystal (16). The autocorrelated pulse width of the dye laser was 8 ps and the cross-correlation frequency was 25 Hz. The dye laser was tuned to the emission maximum of the dye or 590 nm, which yielded 295 nm doubled ultraviolet light for excitation of tryptophan residues. The 295-nm light was passed through a half-wave plate before entering the sample compartment to eliminate any horizontally polarized light. Data were collected with the emission polarizer set at the magic angle of 55” and emission was observed through a 370-nm interference filter (10 nm bandpass). Usually lo-15 different modulation frequencies were used with data collected until phase and modulation deviations reached less than 0.2 and 0.004,respectively. Temperature (25°C) was maintained using a Haake temperature bath; lifetime least-squares analysis was performed using the software supplied by ISS Instruments, Champaign-Urbana, Illinois. For lifetime measurements, cuvettes contained 0.01 M phosphate, pH ‘7.2,0.01% NaN3, and 150 pg of either IL-101or IL18 in a final volume of 2.5 ml (3.33 and 3.47 pM, respectively). A solution of p-terphenyl in cyclohexane (7 = 1.0 ns) was used as the reference lifetime standard.

PI

where F. is the fluorescence of the unquenched protein, F is the fluorescence at quencher concentration [&I, and K., is the Stern Volmer quenching constant. A plot of Fe/F versus [Q] gives a plot with a y intercept of 1 and a slope equal to K,,. Modified Stern Volmer plots were constructed according to

RESULTS

Polyacrylamide Gel Electrophoresis Significantly more disulfide-linked dimer was present in stock solutions of ILla stored at high concentration at either -20 or 4°C then in solutions of IL-l/3 (data not shown). This occurred even though ILl/3 has two cysteine residues and IL-la only one. The tendency to dimerize appeared to be a function of concentration since 0.5 mg/ml solutions had significantly smaller percentages of protein dimer than 8 mg/ml solutions (not shown). However,

FLUORESCENCE

PROPERTIES OF INTERLEUKINS

EMISSION WAVELENGTH(Nm)

la AND lfl

EMISSION WAVELENGTH

FIG. 1. Fluorescence emission spectra of interleukins la and l/3. The fluorescence emission spectra of interleukins lo and 10 was recorded at 25°C with excitation at 275 or 295 nm. IL-la or 18 (100 pg) was dissolved in 2.5 ml 0.01 M sodium phosphate buffer, pH 7.2. Spectra were recorded in the ratio mode as described under Materials and Methods under the conditions below. (A) Interleukin l@ (- - - -) IL-18 exe 275 nm; (- - -) IL-l/3 exe 275 nm + 6 M guanidine hydrochloride; (---) IL-l& exe 295; (- - -) IL-10 + 6 M guanidine hydrochloride, exe 295; (-) IL-l@ + 0.3 M KCl, exe 295. (B) Interleukin lo: (- - -) IL-la exe 2’75; (-) IL-la + 6 M guanidine hydrochloride, exe 275; (- - -) IL-lo exe 295; (---) IL-la + 6 M guanidine hydrochloride, exe 295.

even freshly thawed solutions of IL-la contained small amounts of dimer. Extended storage in solution at 4°C exacerbated this problem; therefore, interleukin lct~ solutions were used either freshly thawed or stored dilute (0.5 mg/ml) at 4°C.

cence signal was observed when excited at 275 nm. No effects of NaCl, KCl, or ammonium sulfate were noted. In the presence of 6 M Gdn-HCl, the emission maximum was shifted to 355 nm with an increase in quantum yield as was seen for IL-l&

Spectral Properties

Acrylamide

of Interleukins

The fluorescence emission spectra of ILlcz and IL-lp are shown in Figs. 1A and 1B. When excited at 275 nm, the emission spectrum of IL-l@ exhibits a fluorescence maximum at 343 nm with a shoulder at 308 nm whereas the spectrum in 6 M Gdn-HCI has a maximum at 355 nm with a more pronounced shoulder at 308 nm (Fig. 1A) and an increased quantum yield. However, when the excitation wavelength was 295 nm (to excite only tryptophan), the emission maximum was also at 343 nm, but no shoulder in the spectrum was seen (Fig. 1A). The fluorescence emission intensity of IL-10 was significantly enhanced by 0.3 M KC1 (Fig. 1A) and also by identical concentrations of NaCl and CsC1;chloride binding was not a factor since 0.3 M ammonium sulfate produced similar effects (data not shown). The emission spectra of IL-la are shown in Fig. 1B. An emission maximum of 343 nm was also observed for this protein with excitation at either 275 or 295 nm, but no shoulder at 308 nm was observed and a strongly enhanced fluores-

Quenching

Results of experiments with the diffusional quencher acrylamide are shown in Figs. 2A and 2B. The Stern Volmer plot for IL-la was linear and that for IL-lp curved slightly downward (not shown) with Stern Votmer quenching constants of 6 and 5.85 M , respectively. The modified Stern Volmer plots (Figs. 2A and 2B) were linear with y intercepts of 1, indicating 100% accessibility of the tryptophans of both proteins to acrylamide. A summary of these and all other quenching constants are shown in Table I. Iodide and Cesium Quenching

The results of iodide and cesium quenching of human IL-la are shown in Figs. 3A 3B, respectively. Attempts were made to obtain iodide quenching data for IL-l@ by adding an amount of KC1 to each iodide addition to hold the ionic strength constant at 0.3 M. However, even under these conditions, consistent quenching results (concentration-dependent) were not obtained

EPPS, YEM, AND DEIBEL

0-

50

0’

10 ACRYLAMlDE(M-‘1

ACRYLAMlDE(M-‘)

20

FIG. 2. Acrylamide quenching of interleukins la and 18. Acrylamide quenching of the interleukins 1 was performed as described under Materials and Methods. (A) IL-la; (B) IL-l@.

for IL-l& The Stern Volmer plot for iodide quenching of IL-la curves downward (not shown) and the modified Stern Volmer plot was linear with an intercept of 2.95 (Fig. 3A). The Stern Volmer quenching constant (5.9 M-‘) was calculated from this modified Stern Volmer plot and the constant value and fractional accessibility to iodide are given in Table I. The ability of cesium, a cationic agent to quench the tryptophan fluorescence of ILlcu is shown in Fig. 3B. Cesium quenching results were similar to those shown for iodide in Fig. 3A. The Stern Volmer plot also curves downward (not shown) as was observed for iodide quenching, and the modified Stern Volmer plot is linear with an intercept of 2.8. The Stern Volmer constant (4.36 M-r) and fractional accessibility to quencher are shown in Table I. We ob-

served no quenching by cesium of IL-l@ fluorescence, even in the presence of KC1 to maintain ionic strength. All additions of cesium increased the fluorescence of IL-l@, as cited earlier in Results. The downward curvature of the Stern Volmer plot is common for proteins which contain more than one class of tryptophans, and suggests that there is a fraction of the total emission which is not accessible to quencher. Fluorescence Lifetimes Multifrequency fluorescence intensity decays of the intrinsic tryptophan fluorescence of interleukins lcu and l@ are shown in Fig. 4 and the parameters are given in Table II. Clearly, the intensity decays of the proteins are well described by a two-exponential decay (Table II); fits

TABLE I FLUORESCENCE QUENCHING CONSTANTS OF INTERLEUKIN 1 PROTEINS KY

acrylamide (Mm’) IL-la IL-16

6 5.85

K” CS

F, acrylamide

(M-l)

1

4.36

1

-

KS” CS

iodide (M-l)

F, iodide

0.36

5.9

0.34

-

-

-

Note. Quenching constants for IL-la and IL-l/3 were calculated from the slope of the modified Stern Volmer plots and the fractional accessibility from the y intercept.

FLUORESCENCE

PROPERTIES

OF INTERLEUKINS

la AND

87

l/3

zo- 6

IO- A 9-

o15-

2l0

5

10

, 15

5

10

KI (M-')

15

CsCl (M-')

FIG. 3. Iodide and cesium quenching of interleukin lo. The effect of ionic quenchers on the intrinsic tryptophan fluorescence of IL-la was studied as described under Materials and Methods (A) Iodide quenching; (B) cesium quenching.

to single- or triple-exponential decays yielded poorer fits with one component having a small fvalue with high standard deviation. The tryptophan lifetime of ILl/3 is characterized by a long component of nearly 5 ns which contributes 88% to the integrated intensities but represents only 44% of the decay. The short component of 0.54 ns, however, contributes only 12% to the integrated intensity but represents

56% of the decay. In the presence of 0.3 M KCI, lifetime values, intensities, and decay contributions are similar to those in the absence of salt. In the presence of a denaturing concentration of guanidine hydrochloride, an average lifetime of 2.6 ns was determined, which is typical for tryptophans in unfolded proteins (19). In contrast, the fluorescent lifetime decay for IL-la (which contains two trypto-

.l go-

6

60706050 4030zoloo- _--- 3 1

.' CJ ' 3 3 '-8,' 10

1 100

.I,.

FREOUENCY (MHZ)

FIG. 4. Frequency dependent intensity decays for IL-la and IL-l@ Multifrequency phase and modulation intensity decays were obtained for IL-lo (A) and IL-l@ (B) as described under Materials and Methods. Individual parameters are given in Table I.

-0

88

EPPS, YEM,

phans) is dominated by a 2.6-ns component which contributes 61% to the integrated intensities but represents only 34% of the decay. The short component of 0.88 ns contributes 39% to the integrated intensity but represents 66% of the decay. Thus, the average lifetime of IL-la (1.93 ns) is much shorter than that for IL-l/3 (4.4 ns). As was the case of IL-l@, the presence of 0.3 M KC1 had little, if any, effect on the lifetime values of IL-la, and the average lifetime (2.41 ns) in the presence of 6 N Gdn-HCl, was similar to that for IL-la under the same conditions. DISCUSSION

Fluorescence spectroscopy is a sensitive technique which can be employed to obtain useful information about protein structure. The purpose of the work presented here was to compare and contrast the tryptophan environments of interleukins lcu and l/3. These cytokines have similar biological activity but very little (25%) sequence homology. Both cytokines contain tyrosine, tryptophan, and free sulfhydryls in the form of cysteine residues. The intrinsic fluorescence of tyrosine and tryptophan residues has been extensively utilized to probe the environments of these fluorophores and to gain some insight into the overall structure of proteins, in particular proteins with similar structures or biological activity. The emission spectra of the two cytokines (Figs. 1A and 1B) provide some information as to the overall nature of the environments of the intrinsic fluorophores in the proteins. A shoulder corresponding to tyrosine fluorescence was seen when interleukin 18, in the native or denatured state, was excited at 275 nm (Fig. 1A). No such shoulder was seen in the comparable spectrum of interleukin la. Since there are three more tyrosine residues in interleukin lcu than in l@, those in the latter protein must be less internally quenched, whether by neighboring groups or by energy transfer to tryptophan, since no tyrosine fluorescence was observed for IL-la. Due to the significant increase in tryptophan emission intensity of IL-la when excited at 275 nm, we hypothesize that tyrosoine

AND

DEIBEL

fluorescence in this protein is lost via energy transfer to tryptophan. The emission spectrum of IL-l@ is shifted to 353 nm under denaturing conditions (6 M Gdn-HCl, Fig. 1A) with a concomitant increase in fluorescence intensity, which may result from the increase in ionic strength rather than unfolding. The emission spectrum of IL-1P also exhibited a significant increase in fluorescence intensity when the spectrum was recorded in the presence of 0.3 M KCl; this phenomenon was not observed for IL-la under identical conditions (data not shown). These results for IL-la are in excellent agreement with data published previously for this protein (11). The emission maximum of IL-la! was also observed to occur at 343 nm, and was likewise shifted to 353 nm in the presence of 6 M guanidine (Fig. 2B), also with an increase in fluorescence intensity. To our knowledge this is the first characterization of the fluorescence properties of IL-la. The increase in the fluorescence intensity of IL-lp resulting from an increase in ionic strength (Fig. 1A) warranted further investigation. It was conceivable that the effect of ionic strength could have been a function of the atomic radius of the salt. Therefore, the effects of three different univalent salts on the intrinsic fluorescence of IL-l/? were studied. The three univalent salts (Na, K, and Cs) were chosen to encompass a range of atomic radii. All three salts produced a similar dose-dependent increase in the fluorescence intensity in the tryptophan fluorescence of IL-l/3 (data not shown). The phenomenon was not a result of chloride binding to the protein since ammonium sulfate produced similar results (data not shown). There are three possible explanations for the ionic strength effects: (i) the addition of salt, and hence increasing ionic strength, results in a conformational change in IL-la secondary and/or tertiary structure such that some of the internal quenching of the tryptophan residue is relieved; or (ii) addition of KC1 results in charge neutralization which relieves static quenching of the tryptophan fluorescence; or (iii) a combination of (i) and (ii). We favor hypothesis (ii) since no shift in the emission maximum in the presence of salt was observed and

FLUORESCENCE

PROPERTIES

OF INTERLEUKINS

TABLE

lo AND

89

10

II

DECAY TIMES OF TRYPTOPHAN EMISSION FROM INTERLEUKINS la AND 10" Sample

71b

SD”

(~1

fi

72

SD

IL-10 no salt IL-lb + 0.3 M KC1 IL-1p +6~ Gdn-HCI IL-la no salt IL-la + 0.3 M KC1 IL-la +6~ Gdn-HCI

4.96

0.04

0.44

0.88

0.54

0.04

5.1

0.05

0.48

0.88

0.63

3.1

0.07

0.53

0.76

2.6

0.08

0.34

2.7

0.09

2.9

0.21

fi

x2R

T a@

0.56

0.12

2.1

4.4

0.08

0.52

0.12

1.4

4.6

1.1

0.07

0.48

0.24

2.9

2.6

0.61

0.88

0.04

0.66

0.39

2.4

1.9

0.34

0.61

0.9

0.04

0.66

0.39

3.8

2

0.48

0.75

0.81

0.3

0.52

0.25

6.3

2.4

a Phosphate, 0.01 M, pH 7.2; 7 = 25°C + added components as b The best fit of the data was obtained for a two-component or three-component fits were much larger, as were the standard ’ Calculated from the diagonal of the covariance matrix. d The average lifetime was calculated from the contributions sponding fractions.

multifrequency lifetime results suggested no effect of salt, since the tryptophan fluorescence lifetime of IL-10 was the same in the absence or presence of salt. This will be discussed later in more detail. If a conformational change occurs as a result of increasing the ionic strength of the medium, this might be reflected in a change in the lifetime values. However, we found identical lifetime values at all concentrations of KC1 up to 0.3 M. Thus we favor the charge neutralization hypothesis although we cannot completely rule out hypothesis (i). Previous studies suggested that the tryptophanyl fluorescence is quenched by a neighboring carboxyl residue with a pK, of approximately 6.59 (11). Publication of the X-ray coordinates, not given in the designated reference, will be necessary to confirm this (18). Alternatively, we cannot rule out the possibility of some form of aggregation which increases at higher ionic strengths. Fluorescence quenching has been utilized to delineate tryptophan environments in a number of proteins (14). The re-

(~2

indicated. lifetime for both proteins; deviations. of the individual

x2R values for one-

components

using the corre-

sults can be used to determine the solvent accessibility of the tryptophan(s), and when ionic quenchers are used, the possible presence of a charged residue(s) in the protein which affects accessibility to the tryptophan(s). Ionic quenchers generally quench surface-exposed tryptophans although it is possible that a tryptophan within a cavity or channel may also be accessible to these agents. Previous work (ll), using solvent perturbation, suggested that tryptophan 120 of IL-lp is 40% exposed to the aqueous environment. Acrylamide quenching results presented here for IL-la and IL-lp indicate that the tryptophan residues in both proteins are totally accessible to the quencher, as evidenced by the modified Stern Volmer plots (Figs. 2A and 2B). The Stern Volmer constants were essentially identical for both interleukins as was the fractional accessibility (lOO%, see Table I). However, when the charged collisional quenchers iodide and cesium were used, very different results were obtained. Although modified Stern Volmer plots of iodide and cesium

90

EPPS, YEM, AND DEIBEL

quenching for IL-la indicated nearly equal accessibility to each agent (34% for iodide and 36% for cesium), Stern Volmer quenching constants were somewhat different (Table I). If one assumes that these accessibilities are a function of solvent exposure, and, allowing for some exclusion of ionic quenchers by ionic groups in proximity to the tryptophans, then these data suggest that solvent exposure of IL-la tryptophans is comparable to that in IL-l@ (40%; Ref. (11)). The quenching constants for iodide and cesium were 5.9 and 4.4 M-', respectively. We attempted to obtain both iodide and cesium quenching data for ILlp by holding the ionic strength constant at 0.3 M with KCl. Under these conditions, there was no change in the fluorescence intensity at all concentrations of quencher. When the same experiments were performed with iodide as the quenching agent, some reduction in tryptophan fluorescence occurred, but was not strictly concentration-dependent. We hypothesize that two competing processes occur when ionic quenching agents are added to solutions of IL-l@: (i) fluorescence enhancement resulting from neutralization of a negatively charged residue which quenches the intrinsic fluorescence, and (ii) the dynamic quenching reaction itself. Thus reduction of fluorescence is more than cancelled out by the effect of ionic strength and hence, no quenching of IL-l/3 tryptophan fluorescence by ionic quenchers was observed. These hypotheses are rigorous for cesium chloride but iodide is a better quencher of IL-l/3 fluorescence leading to complicated interpretations. Fluorescence lifetime experiments with interleukins la and l/3 provided more evidence of differences in the tryptophan environments of the two proteins (Figs. 4A and 4B and Table II). The intensity decay of IL-la is characterized by short and long components of 7 = 0.5 and 5 ns, respectively. The long component dominates the intensity cf = 87.7%). These lifetime data were not significantly altered by the presence of salt (0.3 M KCI), which again suggests that the effect of salt on the tryptophan emission intensity of IL-lp was charge neutralization which relieved static quenching of the fluorescence. The inten-

sity decay of IL-la, a two tryptophan protein, is also characterized by short (T = 0.88 ns) and long (7 = 2.615) lifetime components; however, the long component contributes less (f = 61%) to the total intensity than was the case for IL-l@. Again, we conclude that the presence of 0.3 M KC1 had no effect. If one compares the average lifetime for IL-l@ (7 = 4.4 ns) with that of ILla (7 = 1.93 ns), it is again evident that the single tryptophan of IL-lb resides in a clearly different environment than the two tryptophans of IL-la. Since IL-la is a two tryptophan protein, it is not possible, from the lifetime data alone, to differentiate individual environments for the two residues. It will be necessary to perform lifetime experiments at different emission wavelengths to do these assignments. However, it is evident that the tryptophans of the two proteins, both of which are composed of two-component lifetimes, are in very different environments, even though they have identical fluorescence emission maxima. In summary, we have shown both similarities and differences in the tryptophan environments of interleukins la and lb. These two cytokines have similar biological activities, are presumed to bind to the same receptors, and yet have only 25% sequence homology. It is known from CD experiments that IL-lp and IL-la (W. C. Krueger, unpublished results) contain little, if any a-helical structure and are primarily P-sheet proteins. But since the two cytokines have similar biological activities, the three-dimensional structure (at least the receptor binding region) of the proteins must be similar. In the case of ILlfi, the single tryptophan (position 120) has an emission maximum of 343 nm, total accessibility to penetrating quencher acrylamide, and sensitivity to variations in the ionic strength of the medium which undoubtedly explains our inability to obtain iodide or cesium quenching data, and a lifetime decay characterized by both long and short components. The two tryptophans of IL-la (positions 113 and 139) also have an emission maximum of 343 nm, are totally accessible to the penetrating quencher acrylamide, have similar accessibility to both iodide and cesium, and a life-

FLUORESCENCE

PROPERTIES

time decay also composed of a long and a short component, although the average lifetime is much shorter than that of ILl/3. In addition, IL-la is much more prone to form disulfide dimers, especially at high protein concentrations. This may be a function of the relative exposure of the cysteines to the aqueous environment. Thus, although the two proteins have similar emission maxima, the immediate tryptophan environments appear to be quite different in terms of susceptibility to ionic strength and ionic quenching agents. This is a function of the primary sequence since IL-la: is more highly charged than IL-l/3. The isoelectric points of IL-lp and IL-la are 6.7 and 5.2, respectively (data not shown). Thus the effect of ionic strength on the tryptophan emission intensity of IL-l@ may involve only charged residues in the vicinity of the tryptophan. The fluorescence lifetime data for the two proteins support to our hypothesis that the fluorescence of Trp 120 of IL-l/3 is statically quenched (as compared with Trp 113 and Trp 139 of IL-la) by neighboring charged groups since the decays are identical in the absence and presence of 0.3 M KCl. It will be necessary to further resolve the contributions of each tryptophan of IL-la to the total emission before more direct comparisons can be made.

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OF INTERLEUKINS

la AND

10

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