Journal of Protein Chemistry, Vol. 12, No. 4, 1993

Conformation of the Abortifacient Protein Pinellin: A Circular Dichroic Study

Zong Jin Tao, 1 Zhi Min Shen, 2 and Jen Tsi Yang z'3

Received February 9, 1993

The conformation of pinellin was studied by circular dichroism, which showed a minimum at 223 nm and a double maximum at 198-200 nm. The protein was rich in 3-sheet (about 40%) with little a-helix, based on current CD analyses. It was stable between pH 4 and 10 beyond which it unfolded reversibly, but in alkaline solution, prolongly stored at, say, pH 12, it became irreversibly denatured. Thermal denaturation indicated a transition between 55 ° and 68°C; the solution at 80°C was partially renatured upon air-cooling back to room tem- perature. Addition of sodium dodecyl sulfate caused a sharp increase in a-helix, which leveled off at 0.25 mM surfactant.

KEY WORDS: Abortifacient protein; pinellin; circuiar dichroism; conformation

1. INTRODUCTION 4

Pinellin, a plant protein, is isolated from the juice of the Chinese medicinal herb pinellia (Pinell ia ternata, Breit.) (Tao et al., 1981). It has a molecular weight of 48,000 by sedimentation equilibrium. Pinellin has been crystallized but as yet no x-ray diffraction data are available, nor has its amino acid sequence been completed. Pinellin can be used as an abortifacient protein for terminating early pregnancy of mice (Tao et al., 1981). Its specific binding sites on the uterus or embryos are localized on the epithelium of endometrium and glandula based on in vitro exami- nation (Tao et al., 1983; Chen et al., 1984). Pinellin exhibits cell agglutinin and mitogenic activity that is species-specific. The evidence from hapten inhibition of its hemagglutination of many carbohydrates and glycoproteins suggests that pinellin binds only to mannose and the binding sites are larger than the size of a monosaccharide (Sun et al., 1983). Pinellin

1 Shanghai Brain Research Institute, Academia Sinica, Shanghai 200031, China.

2 Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130.

3 To whom all correspondence should be addressed. 4 Abbreviations: CD, circular dichroism; con A, concanavalin A;

Gu.HC1, guanidine hydrochloride; NaDodSO4, sodium dodecyl sulfate; FPLC, fast pressure liquid chromatography.

has also been used as a marker protein for studying dissociated cultured neurons (Tao et al., 1991).

We have now further purified pinellin by FPLC and studied its conformation by CD, which is most sensitive to conformation and conformational changes of proteins. We report herein the CD results of pinellin in solutions and its stability against extreme pHs, Gu-HC1, NaDodSO4, and thermal denaturation.

387

2. MATERIALS AND M E T H O D S

2.1. Materials

Pinellin was prepared from fresh root tubers of pinellia by ammonium sulfate precipitation and crys- tallized in phosphate buffer (ionic strength 0.1, pH6.8) at 28-30°C. The preparation was further purified by FPLC. All chemicals were of analytical grade. M O N O Q anion exchanger, PD-10, and Superose 12 columns were purchased from Pharrna- cia, and HiPore C4 column was from Bio-Rad.

For pH study, one part of a stock solution of pinellin and nine parts of a buffer were mixed. Buf- fers of constant ionic strength contained glycine for pH 2-3 and 9-12, acetate for pH 4-6, and phosphate for pH 7-8. For denaturation studies, 8 M Gu-HC1

0277-8033/93/0800-0387507.00/0 © 1993 Plenum Publishing Corporation

388 Tao et al.

was added to a protein solution to the desired con- centrations of both protein and Gu-HC1. Likewise, a stock solution of pinellin was mixed with an appro- priate amount of 50 m M NaDodSO4 and diluted with water to the final concentration of 1 - 1 0 m M N a - DodSO 4.

2.2. Circular Dichroism

CD spectra were measured on a JASCO J500A spectropolarimeter with a DP500 data processor for data acquisition and an IBM PC personal computer for data analysis. The instrument was flushed con- stantly with dry nitrogen and the temperature of the solution was controlled by a jacketed aluminium cell holder connected to a Hakke constant-temperature water bath. The CD data were expressed as mean residue ellipticity, [0] in degcmZ/dmol, according to the equation: [0] = (d x s x M o ) / ( c × l), where d denotes observed ellipticity (displacement in cm from the baseline), s sensitivity in mdeg/cm, M0 mean residue weight (115 for pinellin), c protein con- centration in mg/ml, and l light path of the cell in mm. The data of CD spectrum between 190 and 240 nm were analyzed by the methods of Chang et

al., (1978) and Provencher and G16ckner (1981).

2.3. Amino Acid Analysis

Protein samples were hydrolyzed with 5.7 N HC1 for 24h at l l0°C. The acid-free hydrolysates were analyzed on a Beckman 6300 amino acid analyzer at the Depar tment of Biochemistry and Biophysics, University of California at Davis.

0.40 E c

O 0 0 ¢ q

0.20

0

0.00 i 20 40 60

Time, rain

Fig. 1. Rechromatography of pinellin by FPLC on a MONO Q column. The column (HR 5/5) was equilibrated with 0.02M ethanolamine-HC1 buffer (pH9.18) and eluted with a linear gradient of increasing NaC1 concentrations.

Table I. Amino Acid Composition of Pinellin a

Amino acid Number of residues

Asx 69.0 (69) Thr 19.2 (19) Ser 32.2 (32) Glx 35.0 (35) Pro 14.2 (14) Gly 55.2 (55) Ala 15.5 (16) Cys 2.0 (2) Val 29.5 (30) Met 5.0 (5) Ile 19.5 (20) Leu 41.7 (42) Tyr 14.5 (15) Phe 19.1 (19) His 14.5 (15) Lys 20.0 (20) Arg 22.0 (22) Trp n.d.

Total 430

aNumbers in parentheses are rounded to the nearest integer.

3. RESULTS

3.1. Purification of Pinellin

The solution of crystallized pinellin was applied to a M O N O Q column that had been equilibrated with 0.02M ethanolamine-HC1 buffer (pH9.18). The column was eluated by NaC1 solutions of increasing concentration. The fractions of the first peak were pooled and rechromatographed on the M O N O Q column after changing to the starting buffer on a PD-10 column. The purified protein showed little impurities (Fig. 1). It was homogeneous by gel permeation chromatography on Superose 12 column, RP-HPLC on HiPore C4 column, and NaDodSO4-polyacrylamide gel electrophoresis (or SDS-PAGE).

3.2. Amino Acid Composition

Amino acid analysis of pinellin (Table I) indi- cated that it was rich in Asx (Asp +Asn) , and Glx

Table II. Conformational Parameters of Pinellin

Chang ~ Provencher and Fractions b et al. G16chner a

f// 16 3 f~ 41 39 f 21 21 fn 22 37

a Methods: Chang et al. (1978), Provencher and G16ckner (1981). b Fractions: H, a-helix;/3,/3-sheet; t,/3-turn; R, unordered form.

Conformation of Pinellin 389

E 7O

0

4000

Io)

-o -4ooo

-8000 180

100

50

0

-50

I I I -100 200 220 240

~ , nm

I I I I

25( 290

(Glu + Gin), which accounted for 24 mol percent of the 430 residues, and it had two Cys residues. The results of SDS-PAGE indicated that the electrophor- etogram was identical in the presence and absence of mercaptoethanol, suggesting the absence of any inter- chain disulfide bond.

3.3. Protein Conformation

CD spectrum of pinellin in the far-UV region (Fig. 2, left; see also Xu and Lu, 1981) shows a mini- mum at 223nm with a shoulder near 212nm and a maximum at 198-200 nm (the splitting of this posi- tive band is not clear). Together with the rather small magnitudes of the CD bands, the spectrum suggested a #-rich protein with some a-helix. This was sup- ported by CD data analyses by both the methods of Chang et al., (1978) and Provencher and G16ckner (1981) (Table II). The estimates of #-sheet by the two methods agreed well with each other. The esti- mates of a-helix were low, although there was some discrepancy between the two methods.

The near-UV CD bands (Fig. 2, right) can largely be attributed to the chromophores of aro- matic side groups. The positive bands at 293 and 286 nm could be assigned to Trp residues and those at 276, 270, and 266nm were mostly due to Tyr residues.

3.4. pH Effect

Pinellin was stable between p H 4 and 10 (Fig. 3A). The magnitude of [0]222, which corresponded to the broad minimum in the far-UV CD spectrum (Fig. 4A, curve 2), gradually decreased at extreme pHs. The band position was blue-shifted from 223nm at p H 7 to 218nm at p i l l 2 (curve 3) and toward 213nm at p H 2 (curve 1). The denaturation was essentially reversible merely by adjusting the pH

I

330

Fig. 2. CD spectrum of pinellin in aqueous solution (pH7.0) at 23.5°C. Protein concentration: 0.085mg/ml. Cell length: 1 mm for far-UV spectrum (left); 10mm for near-UV spectrum (right).

of the solution to neutral, but the solution at pH 12 was irreversibly denatured after overnight storing.

3.5. Thermal Denaturation

Pinellin began to denature upon raising the tem- perature of the solution above 40°C and its magni- tude of [01210 reached a plateau around 80°C (Fig. 3B). A conformational transition occurred between 55 ° and 68°C. The CD minimum of pinellin was blue-shifted from 223 nm at 25°C (Fig. 4B, curve 1) oo[

- 8 0 4 8 12

pH

1 0 SlO 510 7 0

Temperature, °C

0 2 4 6

Guanidine. HCI, M

I

9O

c

"';~0 4 8 12

NaDodSo 4, mM

Fig. 3. CD of pinellin as a function of (A) pH, (B) temperature, (C) guanidin-HC1 concentration, and (D) NaDodSO 4 concentration.

390 Tao et aL

i e A

= 190 210 230

~ 3

I r I

180 200 220 240

n m

i

250

Fig. 4. CD spectra of pinellin: (A) At different pHs. Curves: I, 2; 2, 7; 3, 12. (B) At various temperatures (°C). Curves: 1, 25; 2, 64; 3, 82; 4, room temperature after cooling back from 82°C.

to about 215 nm at 64°C (curve 2). The CD spectrum at 82°C resembled that of unordered form with a minimum near 200nm (curve 3). It was almost restored to that at the transition temperature (64°C) (curve 4) when the solution was air-cooled back to room temperature, indicating that the thermal dena- turation was partially reversed.

3.6. Guanidine.HC1 Denaturation

Based o n [0]223 rim, pinellin began to unfold in 2 M Gu-HC1, and the magnitude of CD increased sharply up to 3 M Gu-HC1 and approached a plateau around 6 M Gu-HC1 (Fig. 3C). The transition point

A

- 200 220 240

1

i i i

180 200 220 240

n m

Fig. 5. CD spectra of pinellin: (A) In guanidine-HC1 solutions. Curves: 1, zero; 3, 3M; 4, 6M; 2, after removal of 6MGu-HCt through dialysis. (B) In NaDodSO4 solutions. Curves: 1, 0 M; 2, lmM; 3, 1.SmM; 4, 2mM.

occurred at 2.5 M, which agreed with the previously reported value of 2.75 M (Xu et al., 1981). The CD spectrum of pinellin (Fig. 5A) indicated that the pro- tein was essentially denatured in 3 M Gu.HC1 (curve 3) and completely unfolded in 6 M Gu.HC1 (curve 4) (the data below 210 nm were less accurate because of strong absorption of the denaturant). Removal of Gu.HC1 through dialysis restored the spectrum (curve 2) to that of native pinellin (curve 1).

3.7. Effect of Sodium Dodecyl Sulfate

The binding of NaDodSO4 to pinellin drastically altered protein conformation. Based o n [0]208 , as little as 1 mM surfactant would sharply increase the mag- nitude of CD and the process was complete at 2.5 mM NaDodSO4 (Fig. 3D). The broad minimum at 223 nm of the CD spectrum (Fig. 5B, curve 1) also blue-shifted to about 210nm in l mMNaDodSO4 (curve 2). However, unlike acid and alkaline, thermal and G.HC1 denaturation, NaDodSO4 induced the formation of s-helix, as evidenced by the appearance of a double minimum in 1.5mMNaDodSO4 (curve 3). The magnitude of CD spectrum further increased in 2.5mMNaDodSO4 and the negative band was located near 204mm with a shoulder around 220 nm (curve 4), suggesting the presence of partial o~-helix. As expected the process was irreversible upon lowering the concentration of NaDodSO4 from, say, 2.5 mM to i mM because of strong affinity of the surfactant toward the protein.

4. DISCUSSION

CD is probably the most sensitive physical tech- nique for studying protein conformation, but it can- not determine the three-dimensional structure of a protein as the powerful x-ray crystallography and 2D-nmr for proteins in solution do. However, not all proteins can be suitably crystallized for x-ray dif- fraction study and proteins of high molecular weights (over 10,000) are still usually beyond the reach of nmr. CD has two attractive features--the use of a small sample (1 mg or less for far-UV CD measure- ments) and the ease of its operation. It is particularly simple for monitoring conformational changes such as protein denaturation, as is evidenced from the results in this work.

CD data of proteins have been widely used to estimate their various conformations in the protein molecule, but current methods are still empirical and cannot recognize a failed analysis without know-

Conformation of Pinellin 391

ledge of the protein structure (for a review, see Yang et al., 1986). Both the methods of Chang et al., (1978) and Provencher and G16ckner (1981) suggested that pinellin was rich in 13-sheet, but there was some dis- crepancy in the a-helix estimates (Table I). Perhaps it is useful to compare pinellin with another fl-rich pro- tein con A whose CD spectrum also has a minimum at 223nm and a maximum at 197nm, and, further, whose band positions are also red-shifted by about 6 -7 nm from those of regular all-/3 proteins (Wang et

al,, 1992). The magnitudes of CD bands for con A are about twice those of pinellin shown in Fig. 1, suggest- ing that pinellin may have less/3-sheet than con A. X- ray diffraction study of con A indicates that it has 51%/3-sheet, only 2% a-helix, 9%/3-turn, and 37% unordered form (Reeke et al., 1975). Analysis of CD spectrum of con A by the method of Provencher and G16ckner (1981) agrees well with the x-ray results. On the other hand, the method of Chang et al., (1978) overestimates the fraction of a-helix (21%). These findings led us to believe that the 16% a-helix in Table I I may also have been too high, although this tentative conclusion must be proved or disproved when x-ray diffraction study of pinellin becomes available.

Our study indicated that pinellin was a relatively stable protein against denaturation, even though it has no disulfide bond. Extreme pHs (above 10 or below 4) and temperatures above 70°C are necessary for complete unfolding of the protein. Further, dena- turation by 6MGu.C1 could be reversed merely through dialysis to remove the denaturant. Addition of NaDodSO4 would induce the fraction of a-helix at the expense of/7-sheet, as is true for many proteins in NaDodSO4 solution.

Three other proteins isolated f rom Chinese herbs-- t r ichosanthin , a-, and /3-momorchar in-- have also been classified as abortifacient proteins. Their CD spectra are similar to each other; they show a double minimum at 220 and 208 nm and a maximum at 193 nm (Kubota et al., 1986), suggesting the presence of both a-helix and/7-sheet. These three proteins contain 40 -60% /3-sheet and about 30% a-helix, based on the method of Chang et al., (1978),

suggesting that they are also rich in d-sheet. X-ray diffraction study at 4 A resolution of trichosanthin crystals grown from an alkaline solution (pH8.6) indicates a 39% a-helix and 32% t3-sheet (Pan et

al., 1983) (a different crystalline form can be obtained from an acidic solution at p H 5.4). Their similar pharmacological properties are tempting us to suggest that segments of ,8-sheets might have been involved in the binding sites of trichosanthin, a- and/%momorchar in , and pinellin.

ACKNOWLEDGMENT

This work was supported in part by U.S. Public Health Service grant GM-10880-32.

REFERENCES

Chang, C. T., Wu, C.-S. C., and Yang, J. T. (1978). Anal. Biochem. 91, 13-31.

Chen, H. L., Song, J. F., and Tao, Z. J. (1984). Acta Physiol Sinica 36, 388-392.

Kubota, S., Yeung, H. W., and Yang, J. T. (1986). Biochim. Biophys. Acta 871, 101-106.

Pan, K. Z., Zhang, Y. M., Lin, Y. J., Wu, C. W., Zheng, A., Chen, X. Z., Dong, Y. C., Chen, S. Z., Wu, S., Ma, X. Q., Wang, Y. P., Zhang, M. G., Xia, Z. X, Tian, Q. Y., Fan. Z. C., Ni, C. Z., Ma, Y. L., and Sun, X. X. (1985). In Advances in Chinese Medicinal Materials Research (Chang, H. M., Yeung, H. W., and Koo, A., eds.), World Scientific, Singapore, pp. 297-303.

Provencher, S. W., and G16ckner, J. (1981). Biochemistry 20, 566- 570.

Reeke, G. N., Jr., Becker, J. W., and Edelman, G. M. (1975). J. Biol. Chem. 250, 1525-1547.

Sun, C, Xu, J. H., Zhai, S. K., Tao, Z. J., Yau, T. Y., Zhu, Z., and Shen, Z. W. (1983). Acta Biochim. Biophys. Sinica 15, 333- 338.

Tao, Z. J., Xu, Q. Y., Wu, K~ Z., Lian, S. H., and Sun, D. (1981). Aeta Biochim. Biophys. Sinica 13, 77-82.

Tao, Z. J., Chen, H. L., Sun, C., and Song, J. F. (1983). Acta Physiol. Sinica 35, 107-111.

Tao, Z. J., Bao, X., Wang, P. F., and Dou, Y. M. (1991). Proc. Shanghai Neurosci. Soc. 7, 93.

Xu, Q. Y., and Lu, Z. X. (1981). Acta Biochim. Biophys. Sinica 13, 379-384.

Xu, Q. Y., Sun, D., and Tao, Z. J., (1981). Acta Biochim. Biophys. Sinica 13, 153-159.

Wang, J. M., Takeda, A., Yang, J. T., and Wu, C.-S. C. (1992). J. Protein Chem. 11, 157-164.

Yang, J. T., Wu, C.-S. C., and Martinez, H. M. (1986). Methods Enzymol, 130, 208-269.