Journal of Protein Chemistry, Vol. 10, No. 4, 1991

Basic Fibroblast Growth Factor is a p-Rich Protein Chuen-Shang C. Wu, !'3 Stewart A. Thompson, 2 and Jen Tsi Yang 1

Received April 15, 1991

The conformation of the 153-residue form of human basic fibroblast growth factor (bFGF) was studied with circular dichroism (CD) and sequence prediction methods. The ['ar-UV CD spectrum with a minimum at 202 nm resembled that of an unordered polypeptide/protein or a protein rich in distorted antiparallel fl-sheets. Analysis of the CD spectrum by the leastsquares method of Chang et al. (1978) and the CONTIN program of Provencher an d G16ckher (1981) suggested that about one half of the molecule consisted of fl-sheet and there was no a-helix. These estimates agreed with the prediction by the sequence method of Gamier et al. (1978) using decision constants based on CD results, bFGF had an unusual CD band at 187 nm, which disappeared upon ionization of Tyr side chains at pH 11.7. It also had another unusual property of irreversibly converting the CD spectrum to a helix-like one with a double minimum at 205 and 215 and a maximum at 189 nm upon heating the solution to above 55°C. The helicity was also enhanced in trifluoroethanol and in sodium dodecyl sulfate. The mutant bFGF in which cysteines 76 and 94 were replaced by serine residues had essentially the same properties as the wild-type. KEY WORDS: sulfate.

Basic fibroblast g r o w t h factor; circular d i c h r o i s m ; t r i f l u o r o e t h a n o l ; s o d i u m dodecyl

1. INTRODUCTION 4

et al., 1988; Broadley et at., 1989; Klingbeil et aL, 1991). The genes for b F G F have been cloned from amphibian (Kimelman et al., 1988) and mammalian (Abraham et al., 1986) sources and encode for 155residue ( 1 7 k D ) proteins with methionine (ATG) initiation sites. In addition, alternative upstream Leu (CTG) initiation sites encoding 22 kD, 23 kD, and 23.5 kD forms have also been identified (Florkiewicz and Sommer, 1989; Prats et aL, 1989). The 155residue form of the human protein has been expressed in E. coli (Squires e t a [ . , 1988; Iwane et al., 1987; Fox eta[., 1988; Thompson el al., 1991), and a large amount of the recombinant protein can be', produced for structural characterization. Circular dichroism (CD) is a powerful tool for measuring the conformation or conformational changes of proteins in solution. The far-UV CD spectrum, which measures the protein backbone conformation, has been used frequently to calculate the secondary structure of proteins (Greenfield and Fasman, 1969; Chang et al., 1978; Bolotina et al., 1980; Hennessey and Johnson, 1981; Provencher and

Basic fibroblast growth factor which has been purified from numerous sources (Gospodarowicz, 1987) is a 17 kD protein with two distinguishing characteristics. First, it has an unusually strong affinity for heparin as characterized by its elution from heparin-Sepharose at 1.5 M NaCI (Gospodarowicz et al., 1984). Second, it is a potent mitogenic factor for a variety of cells of mesodermal origin (Gospodarowicz et al., 1987; Burgess and Maciag, 1989) and some epidermal cells such as keratinocytes (O'Keefe et al., 1988; Shipley et al., 1989). The protein has angiogenic activity (Folkman and Klagsbrun, 1987) and can promote wound-healing in animal models (Davidson et al., 1985; McGee

Cardiovascular Research Institute, University of California, San Francisco, California 94143-0524. 2 California Biotechnology Inc., 2450 Bayshore Parkway, Mountain View, California 94043. 3 To whom all correspondence should be addressed. 4 Abbreviations used: bFGF, basic fibroblast growth factor; CD, circular dichroism; NaDodSO4, sodium dodecyl sulfate; TFE, 2,2,2-trifluoroethanol; DC, decision constant.

427 0277-8033 91 0800-0427506 5(. 0 ~ 1991 Plenum Publishing c.'tlrporation

428

G16ckner, 1981). The near-UV CD spectrum, on the other hand, measures the conformation of the aromatic side chains and disulfide bridges, and hence the tertiary structure of the protein (Strickland, 1974). Based on CD studies, the 147-residue form of bFGF was thought to have mainly unordered conformation because its far-UV spectrum resembled that of an unordered polypeptide or a denatured protein (Fox et al., 1988). In this report, we reexamined the conformation of human bFGF (Thompson et al., 1990) by CD and sequence prediction methods. In contrast to the report by others that bFGF is unordered in solution (Fox et al., 1988), we found that this protein is rich in /~sheets. Further, there was an unusual CD band below 190 nm, and the thermal transition converted its spectrum from two minima below 202 nm to a helix-like spectrum. The double cysteine mutant of bFGF, in which cysteines 76 and 94 were replaced by serine residues by muta~enesis, behaved similarly.

2. M A T E R I A L S A N D M E T H O D S 2.1. Production and Purification of Recombinant bFGF

The expression and purification of human bFGF were carried out as described previously (Thompson et al., 1991). Briefly, 50 ml of Luria-broth containing 50 #g/ml ampicillin (Sigma) was inoculated with a single colony of E. coli B containing the bFGF expression vector pTsF-9dH3-154 (which produces the 153 amino acid form of the protein representing deletion of the N-terminal Ala) (Thompson et aI., 1991) and grown at 30°C to stationary phase. This overnight culture was then used to inoculate 1 L of supplemented minimal media containing M9 salts (Sambrook et al., 1989) with casamino acids and 50/~g/ml ampicillin, and the culture was incubated with shaking at 30°C until the absorbance of the culture at 550 nm reached 0.5-0.7. Fifty milligrams of 3]~-indoleacrylic acid (Sigma) were then added to the culture to induce production of the protein. The culture was then incubated with shaking for 16-24 h at 30°C. The cells were harvested by centrifugation. The cell pellet from a I L culture was resuspended in 25ml of 20mM sodium phosphate (pH 7.0), 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (Boehringer-Mannheim). Lysozyme (Sigma) was then added to a final concentration of 0.5 mg/ml, and the suspension was incubated on ice for 30 rain. After lysozyme treatment, the cells were

Wu et al.

ruptured by sonication. Bovine pancreatic RNAse A (Sigma) and bovine pancreatic DNAse I (BoehringerMannheim) were added to a final concentration of 1/~g/ml each, and the solution was again incubated for 30 min on ice. The solution was then centrifuged at 29,000 g for 30 min. The pellet was discarded and the supernatant containing the bFGF was retained. SP Sephadex C-25 (Pharmacia) was equilibrated in starting buffer (20mM sodium phosphate at pH 7.0, 5 mM EDTA, and 0.1 M NaC1) at 4°C. The cell lysate was loaded onto the column and the eluate was monitored at 280 nm with an in-line UV detector. After completion of loading, the column was washed with the equilibration buffer until the absorbance of the eluate returned to the baseline level. The eluant was changed to 20 mM sodium phosphate (pH 7.0), 5 mM EDTA, and 0.6 M NaC1, and the peak of eluting material was collected. A heparin Sepharose (Pharmacia) column (2.5 x 2 cm) was equilibrated with 20 mM sodium phosphate (pH 7.0), 5 mM EDTA, and 0.6 M NaCI, and the 0.6 M NaC1 eluate from the SP Sephadex column was loaded onto this column. After loading, the column was washed with 20 mM sodium phosphate (pH 7.0), 5 mM EDTA, and 0.6 M NaC1 until the absorbance of the eluate monitored at 280 nm returned to the baseline level. The recombinant bFGF bound to the column was then eluted with 20 mM sodium phosphate (pH 7.0), 5 mM EDTA, and 2.0 M NaC1. The mutant bFGF in which Ser residues were substituted for the Cys residues at positions 76 and 94 were produced by site-directed mutagenesis and purified the same way.

2.2. Circular Dichroism

CD spectra were measured on a Jasco J-500 automatic recording specropolarimeter coupled with a DP-500 data processor for data acquisition and an IBM PC computer for data analysis. For temperature control, a specially designed aluminum block with a jacket attached to a Hakke constant-temperature regulator served as the cell holder. The temperature of the sample solution was measured by the Bailey thermometer (Model BAT-2, Bailey Instruments Inc.) with a probe inserted to the solution. Both the instrument and cylindrical cells of various path lengths were calibrated with d-10 camphorsulfonic acid (Chen and Yang, 1977). Unless otherwise stated, the CD spectra were generally measured at 25°C, and scanned eight times at a speed of 10 nm/min and signal-averaged.

Basic Fibroblast Growth Factor

429

The CD data were expressed as mean residue ellipticity, [0], in deg cm2/dmol -~, which was calculated according to the equation: [0] = (d x s x M o ) / ( c

x

(1)

l)

where d denotes observed ellipticity (the displacement in cm from the baseline), s sensitivity in mdeg/cm, M0 mean residue weight (111 for bFGF), c protein concentration in mg/ml, and l the cell pathlength in mm. For native bFGF, CD spectra were measured in 20mM sodium phosphate (pH 7.0). To study the effect of temperature, the protein solution was allowed to equilibrate at each temperature level for 40-60 rain before the spectrum was scanned. For experiments in 2,2,2-trifluoroethanol (TFE, Sigma) and sodium dodecyl sulfate (NaDodSO4, Fluka), appropriate amounts of concentrated stock solutions of these reagents were added to the protein solution to desired concentrations and CD spectra were measured immediately after mixing. The CD data in the far-UV region were analyzed by the method of Chang et al., (1978) using the reference spectra obtained from 15 proteins of known structure (Yang et al., 1986), and that of Provencher and Gl6ckner (1981). For bFGF in NaDodSO4 solution, the spectra were also analyzed according to Wu et al., (1981). The secondary structure of bFGF was also predicted from its sequence by the method of Chou and Fasman (Chou and Fasman, 1974; Prevelige and Fasman, 1989) and that of Garnier et al., (1978).

2.3. Other Methods and Materials The number of SH groups in bFGF was determined by the method of Ellman (1959). The protein concentration was measured by the method of Lowry et aI. (1951) using a bFGF standard curve which was calibrated by amino acid analysis.

3. RESULTS 3.1. Far- and Near-UV CD Spectra of Native bFGF The far-UV CD spectrum of the 153-residue bFGF was comparable to that of the 147-residue bFGF (Fox et aL, 1988; Arakawa et al., 1989), both in magnitude and band positions (Fig. 1A). It showed a small extremum at 227 nm (1000 deg cm 2 drool -~) and a large minimum at 202 nm (-8000 deg cm 2 drool-1), thus ruling out an a-helixrich protein. Rather, the spectrum resembled that of an unordered form or a distorted fl-sheet-rich protein such as the soybean trypsin inhibitor (Mori and Jirgenson, 1981). However, there was another minimum at 187 nm (-11,000 deg cm 2 drool-~), which is not present in soybean trypsin inhibitor nor is reported for the 147-residue bFGF (Fox et al., I988; Arakawa et al., 1989). Further, this CD bard is not found in the spectra of the model a-helix or/3-sheets even when their spectra are extended to the vacuumUV region (Brahms and Brahms, 1980).

r "7 O

B

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-8

-12 180

I 200

I 220

I 240

-0.2 220

I

I

[

I

I

240

260

280

300

320

~%, n m Fig, 1. Far-UV (A) and near-UV (B) CD spectra of bFGF at 25°C in 20 mM Na-phosphate, pH 7.0. Protein concentrations were 0.11 mg/mI for far-UV and 0.22 rag/m1 for near-UV spectra.

430

Wu et al.

The near-UV CD spectrum of bFGF (Fig. 1B) reflects the environment of its aromatic side chains (bFGF has 8 Phe, 7 Tyr, and 1 Trp residues). The rather high intensity of the spectrum suggested that bFGF was a compact molecule. The shoulder at 262 and 268 nm could be assigned to the optical activity of Phe and the trough at 276nm to that of Tyr (Strickland, 1974). Although there is one Trp in the molecule, the near-UV CD spectrum did not show any CD band in the 280-290 nm region, suggesting that this side chain may be exposed to the medium (Strickland, 1974). In addition to these aromatic bands, there was a sharp negative band at 243 nm which is also present as a minor band in soybean trypsin inhibitor (Baba et al., 1969). The four cysteines in bFGF were fully reduced, thus excluding their contribution to the CD spectrum in the near-UV region. 3.2. Effect of Ionization of Tyrosyl Side Chains on the CD Spectra of bFGF

Solvent

Method"

20 m M Na-phosphate p H 7.0, 25 ° CWY PG 50 ° CWY PG 55 ° CWY PG 75 ° CWY PG p H 11.7, 25 ° CWY PG Trifluoroethanol 20% CWY 40% CWY 80% CWY 10 m M NaDodSO4 ,oH 7 CWY WIY pH 2 CWY WIY

fit

f,

f~

fR

0 0.05 0 0.09 0.11 0.12 0.12 0.14 0.01 0.10

0.54 0.72 0.53 0.43 0.36 0.34 0.40 0.37 0.40 0.34

0.13 0.21 0.06 0.14 0.08 0 0.08 0.10 0.11 0.24

0.33 0.03 0.41 0.33 0.45 0.54 0.40 0.38 0.48 0.32

0.20 0.30 0.43

0.45 0.25 0.04

0.03 0 0

0.32 0.45 0.53

0.09 0.17 0.18 0.25

0,51

0 -0.09 --

0.40 0.83 0.38 0.75

0,35 --

" C W Y , C h a n g et al. (1978) ; PG, Provencher and Gl6ckner ( 1981 ) ; WIY, W u et al. (1981).

Tyrosine has a negative band at 190 nm and two positive bands at 202 and 227 nm (Brahms and Brahms, 1980). Thus, the possible contribution of tyrosine transitions to the bFGF CD bands at 187, 227, and 243 nm was assessed by following spectral changes upon ionization of tyrosines at alkaline pH. In the far-UV region, as the pH was increased from 7-10.7 and then to 11.7, the 187 band decreased and eventually disappeared, whereas the 227 nm band changed from positive to negative (Fig. 2). At the same time the 202 nm band increased by about 25%

5

Table I. Conformation of Basic Fibroblast Growth Factor by C D Analysis

at pH 10.7 and by about 100% at pH 11.7. This is consistent with the decrease of the three tyrosine bands upon ionization of the side chains. Analysis of the spectrum atpH 11.7 revealed thatf~ decreased but there were no changes infu or f, as compared to those at pH 7 (Table I). In the near-UV region, the magnitude of the entire CD bands decreased as the pH was increased from 7-10.7 and became a single positive band of

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Fig. 2. Effect o f p H on the far-UV and near-UV C D spectra of b F G F at 25°C. Curves 1, p H 7.0: 2, p H 10.7; 3, p H 11.7.

Basic Fibroblast Growth Factor

431

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3. Effect of temperature on the far-UV C D spectrum of b F G F . (A) 0°C; (B) 41°C; (C) 50°C; (D) 55°C; and (E) 75°C. Buffer and protein concentration were the same as in Fig. 1.

247 nm at pH 11.7 (Fig. 2B), which is essentially that of the ionized tyrosine (Baba et al.,1969). 3.3. Effect of Temperature on the CD Spectra of bFGF The far-UV CD spectrum of bFGF at 0°C (Fig. 3A) was indistinguishable from that at 25°C (Fig. 1A, curve 1). However, as the temperature of the sample solution was raised from 25-75°C, dramatic changes occurred. The 187 nm band decreased as the temperature was raised to 41°C (Fig. 3B), disappeared completely at 50°C (Fig. 3C), and became a positive band at 55°C (Fig. 3D). The 202 nm minimum at 0°C became slightly larger and was blue-shifted to 200 nm at 41°C. At 55°C, there was a broad minimum around 205-215 nm (Fig. 3D) which resembled that of an aggregated helix (Wu et al., 1979). However, this broad minimum and the maximum at 189 nm were somewhat blue-shifted as compared to a normal helix (208-222 and 192 nm) or an aggregated helix (210223 and 195nm). Above 55°C, the solution was slightly turbid, indicating that there was some aggregation, but there was little change in the spectrum between 55°C and 75°C (not shown). In another experiment at 75°C, the minimum at 205 nm was larger than that at 215 nm (Fig. 3E). The spectral change was irreversible; decreasing the temperature from 75-25°C did not reverse the spectrum to the original. If the protein was heated at 50°C for 2 hr (Fig. 3C) and left at room temperature for 6 days, it

had a spectrum close to that observed at 55°C (Fig. 3D). The plots of [0]187 and [0]220 vs. temperature (Fig. 4) show that [01187 became more positive and [0]22o more negative with increasing temperature. The mid-point of the transition was at about 49°C. In the near-UV region, the 243 nm band became more negative as the temperature was raised from

1 -

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Temperature, °C Fig. 4. Plot of temperature vs. ellipticity at 187 n m ( O ) and 220 nm ( 0 ) . Experimental conditions were the same as in Fig. 2.

432

W u et al.

25°C (Fig. 5, curve 1) to 50°C (curve 2), but the bands from other aromatic residues remained the same. At 75°C, the 243 nm band was even more negative and the whole near-UV bands melted together into a monotonous hyperbolic curve (curve 3), again, suggesting an aggregation of the protein at a high temperature. 3.4. Effect of TFE on the CD Spectrum of bFGF

Low concentrations of T F E (5-15%) caused b F G F to precipitate. At 20% TFE, the solution became clear and b F G F assumed a fl-form-like spectrum with a minimum at about 213 nm and a maximum at 192 nm (Fig. 6, curve 1). At 40% TFE, the spectrum showed a double minimum at 207 and 222 nm and a maximum at 192 nm, which are characteristic of an a-helix (curve 2). The CD intensity increased further with increasing concentration of TFE and approached a plateau at about 80% TFE (Fig. 6, inset). The fl-to-a transition midpoint occurred at about 30% TFE.

surfactant NaDodSO4 often induces helical conformation in proteins, the extent of which depends on the helix-forming potential of the protein polypeptide chain (Wu et al., 1981; Wu and Yang 1981). In l0 mM NaDodSO4, b F G F formed a partial helix at neutral pH (Fig. 7, curve 1) and the intensity of the spectrum increased slightly at pH 2 (curve 2). This is due to the protonation of acidic groups on the protein which repel the negatively charged NaDodSO4 at neutralpH (Wu et al., 1981). The surfactant at concentrations higher than 10 mM had no further effect on the CD spectrum of bFGF. We also studied the conformation of a b F G F mutant in which serine residues were substituted for cysteine residues at positions 76 and 94. The mutant had full biological activity but no tendency to aggregate oi form intermolecular disulfides. We found that the mutant protein was indistinguishable from the wild-type b F G F in its CD spectra, the behavior on heating, and the conformation in TFE and NaDodSO4.

3.5. Effect of NaDodSO4 on the CD Spectrum of bFGF

2

In contrast to urea and guanidine-HC1 that unfold proteins to unordered structures, the o

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1

I

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200

220

240

X, nm Fig. 6. Effect of trifluoroethanol on the far-UV CD spectrum of bFGF. Curves, 1, 20%; 2, 40%; and 3, 80% trifluoroethanol. Buffer and temperature were the same as in Fig. 1. Protein concentrations were 0.03-0.2 mg/ml. Inset: Plot of ellipticity at 222 nm vs. the concentration of trifluoroethanol.

Basic Fibroblast Growth Factor 10

-

5

-

433 These results contradict the observation that proteins and polypeptides are unlikely to form /3-sheets in NaDodSO4 solution above the critical micelle concentration of the surfactant (Wu and Yang, 1981). When the CD spectra (Fig. 7) was analyzed by the method of Wu et al. (1981) assuming that proteins do not form//-sheets in NaDodSO4 solution, b F G F had 17% and 25% helix at pH 7 and 2, respectively (Table I).

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3.7. Sequence Prediction of bFGF

¢D 03 01

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-5

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I

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Fig. 7. Effect of sodium dodecyl sulfate on the far-UV C D spectrum of b F G F at 25°C. Curves 1, 10 m M NaDodSO4 at p H 7.0; 2, 10 m M NaDodSO4 at p H 2.

3.6. Analysis of CD Spectra The various conformations in bFGF were calculated from the far-UV CD spectra by both the leastsquares method of Chang et al. (1978) and the CONTIN program of Provencher and G16ckner (1981) (Table I). The two methods in general agreed well for the estimations. Notable exception was the much largerfp (and smallerfR) at 25°C by the ProvencherG16ckner method than by the method of Chang et al. (1978). In aqueous solution below 50°C, bFGF was a/3-sheet-rich protein with a trace of a-helix. At 55°C and higher temperatures, the fraction of a-helix was enhanced at the expense of/3-sheet and /3-turn. In TFE/water solutions, the fraction of the helix increased and that of the /3-form decreased with increasing concentration of TFE. At 80% TFE, 43% of the molecule was in a-helical conformation and only a trace of/3-form remained. In NaDodSO4 solution, b F G F had more/3-sheet than a-helix when analyzed by the method of Chang et aL (1978) (Table I).

To complement the CD methods of analysis, the secondary structure of bFGF was also predicted from its sequence by the method of Chou and Fasman (1974) and that of Garnier et aL (1978). The former is widely used because of its simplicity. It predicted a moderate amount of a-helix and p-sheet and one third of the b F G F molecule in/3-turn (Table II, CF). These results markedly differed from the estimated secondary structures based on the CD data. In the Chou and Fasman method (1974), segments of the sequence often have both a-helix and/3-sheet-forming potentials; thus, each amino acid residue is not unambiguously assigned one conformational state, although we found that this was not a serious problem for bFGF. In contrast, the method of Gamier et aL (1978) does not have this ambiguity, but it can give a wide range of predictions depending on the decision constants (DC) used (Table II, GOR0, GOR1, and GOR2). The choice of a correct set of DC requires independent measurements of a-helix and//-sheet, and this is usually achieved from CD. If such data are not available, a preliminary prediction can be made with all DC equal to zero (Table II, GOR0). With the result of GOR0 as a guideline (30% a-helix and 28% p-form), we obtained GOR 1 by choosing DCs for 20-50'70 ahelix and more than 20% /3-form. This resulted in more a-helix and/?-sheet and less/3-turn than those based on the CF method. The results of GOR 1 again

Table II. Sequence-Predicted Secondary Structure of Basic Fibroblast Growth Factor Met hod~

fH

f~

f

f~

CF GOR0 GOR1 GOR2

0.26 0.30 0.34 0.05

0.19 0.28 0.40 0.61

0.34 0.31 0.20 0.23

0.21 0. l 1 0.06 0.l I

" C F , C h o u and F a s m a n (1974) ; G O R 0 , Garnier et al. (1978) using D C H = 0 , D C p = 0 ; G O R I , same as above using D C ~ = - 7 5 , D C ¢ = - 8 7 . 5 ; GOR2, same as above using DCH = 158, D C ¢ = -87.5.

434

Wu et al.

markedly differed from those based on the CD analysis. With the CD results of native bFGF (Table I), we then chose DCs equivalent to less than 20% ahelix and more than 20% fl-form and obtained the results of GOR2 (Table II). The agreement between GOR2 and CD estimates was better than both the CF method and GOR1.

Rather, it resembled that of soybean trypsin inhibitor which belong to the second type of fl-sheetrich proteins (Manavalan and Johnson, 1983). CD analysis by the two methods (Chang et al., 1978; Provencher and G16ckner, 1981), as well as the sequence prediction (Garnier et al., 1978), also agreed that bFGF was a fl-sheet-rich protein. From its CD spectrum, Arakawa and coworkers (1988) suggested that bFGF is predominantly unordered. More recently, however, based on Fourier transform infrared spectroscopy, they arrived at a conclusion that bFGF is indeed a fl-sheet-rich protein (Arakawa et al., 1991, Prestrelski et al., 1991). From its sequence, bFGF was thought to be a member of a new protein family of homologous growth factors that include interleukin-la and l fl

4. DISCUSSION The CD spectrum of bFGF in the 190-240 nm region resembled that of unordered polypeptides or denatured proteins. However, the minimum in the 200 nm region was somewhat red-shifted to 202 nm as compared to that of the unordered polypeptides.

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Fig. 8. Comparison of secondary structures of bFGF (sequence-predicted) and interleukin-lfl (from x-ray). The sequences were aligned according to Thomas and Gimenez-GaUego (1986). *** a-helix; - - ~-sheet; > > > fl-turn.

Basic Fibroblast Growth Factor

(Gimenez-Gallego et al., 1985; Thomas and GimenezGallego, 1986). According to x-ray crystallography (Finzel et al., 1989; Graves et al., 1990), both interleukin-1 a and lfl have 12 antiparallel fl-strands which are folded into an architecture similar to the soybean trypsin inhibitor (McLachlan, 1979). These and our CD and sequence prediction results of b F G F seem to suggest that b F G F may have a similar folding motif as that of interleukin-1 ct and l fl. When the two proteins were alligned according to sequence homology (Thomas and Gimenez-Gallego, 1986), the secondary structure of b F G F predicted by G O R 2 agreed quite well with that of interleukin-lfl solved by x-ray (Fig. 8). However, the CD spectrum of interleukin-lfl with a weak double minimum at 205 and 214 nm and a maximum of 197 nm (Craig et al., 1987) shows little resemblance to that of b F G F (Fig. 1A). Thus, in spite of the primary and secondary structural homologies, the possibility that the two proteins may have different three-dimensional structures cannot be excluded. The 187-nm band of b F G F in the far-UV region could not be attributed to any known secondary structures. However, our p H study suggested that it may be due to tyrosine side chains. In addition to their characteristic near-UV CD spectra, aromatic amino acids also possess CD bands in the far-UV region (Strickland, 1974; Brahms and Brahms, 1980). These CD bands may affect the backbone spectra of the protein when the content of these amino acids are high and when they are in a rigid environment and in close proximity. In b F G F , the absence of Trp CD band in the near-UV region (Fig. 1B), together with its quenched Trp fluorescence (Arakawa et al., 1989), suggests that this amino acid is exposed and has little effect on the far-UV CD signal of b F G F . However, 3 out of 8 phenylalanine residues are clustered at positions 100-102, 2 at positions 37 and 38, and 4 out of 7 tyrosine residues are located between positions 110 and 122. W-hether these side chains are clustered together in the three-dimensional structure of b F G F could not be determined at present, but their contributions to the far-UV CD spectrum of b F G F may not be ignored. The fact that the ionization of tyrosine at p H 11.7 abolished the 187 nm-band of b F G F strongly suggests that this CD band may be due to tyrosine. At present, except in a few laboratories where the vacuum CD instruments are available, most CD measurements are restricted to wavelengths above 190 or 200 nm and may have failed to detect this unusual band in many proteins. As the instrumentation improves, more information below 190nm may become available.

435

The other unusual feature of b F G F was that heating induced a transition from a spectrum with two minima at 187 and 202 nm to a helix-like spectrum. In general, most aggregated proteins have a distorted double minimum ([0]208 < [0]222), or in extreme cases, a fl-like spectrum (Wu et al., 1979). There were some aggregates in b F G F above 55°C, but the spectrum showed only little (Fig. 2D) or no distortion (Fig. 2E), except that the double minimum and the maximum of the heated b F G F were blue-shifted by a few nanometers as compared to those of the regular ahelix. It is not known whether this spectrum truly represented a-helix, but CD analysis revealed that either fl-form or fl-turn in native b F G F was converted to 10-15% a-helix in heated b F G F (Table I). This phenomenon could not be explained at present. In conclusion, both CD analysis and the sequence prediction method of G a m i e r et al. (1978) revealed that b F G F is not an unordered but a fl-sheetrich protein. In spite of their sequence and predicted structural homology, b F G F and interleukin-lfl have very different CD spectra. Whether their three-dimensional structures have any resemblance has to await for future investigation. ACKNOWLEDGMENT The work was supported by U.S. Public Health Service grant GM-10880-31 (C.S.C.W. and J.T.Y.) and SBIR grant H L 39348-03 (S.A.T.)o REFERENCES

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