Trichosanthin’s interfacial interactions with phospholipids: a monolayer study

Colloids and Surfaces B: Biointerfaces 39 (2004) 105–112

Trichosanthin’s interfacial interactions with phospholipids: a monolayer study Xiao-Feng Xia a , Fu Wang a , Mengsu Yang b , Sen-Fang Sui a,∗ a

b

Department of Biological Sciences and Biotechnology, State-Key Laboratory of Biomembrane, Tsinghua University, Beijing 100084, China Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China Available online 3 March 2004

Abstract Lipid monolayer at the air/water interface, as half a membrane, was used here to investigate the interaction between trichosanthin (TCS), a ribosome inactivating protein, and phospholipid membrane. First, the protein adsorption experiments showed that the negatively charged DPPG caused obvious enrichment of TCS beneath the monolayer, indicating electrostatic attraction between TCS and the negatively charged phospholipid. Second, when TCS was incorporated into the phospholipid monolayer, it could not be completely squeezed out until the monolayer collapsed. The results were demonstrated to be irrelative with the phospholipid headgroup, suggesting a strong hydrophobic force between TCS and phospholipid hydrocarbon chain was involved in the interaction. Third, the protein/membrane interaction was further studied with fluorescence microscope. The results showed that TCS could penetrate into both the condensed and the fluid phase of the DPPG monolayer under low pH condition and eventually resulted in a homogeneous phospholipid phase. The breakage of ordered packing of phospholipid by TCS may be responsible for this homogenizing effect. © 2004 Elsevier B.V. All rights reserved. Keywords: Lipid/protein interaction; Air/water interface; π–A isotherm; Epifluorescence microscopy

1. Introduction Trichosanthin (TCS) is the active component isolated from a Chinese herbal medicine Tianhuafen (the root tuber of Trichosanthes kirilowii maxim, Cucurbitaceae) [1,2]. It has long been used clinically in China to terminate early and midtrimester pregnancies [3] and to treat trophoblastic tumors [4]. Recent studies have revealed a broad spectrum of other biological and pharmacological properties of TCS, including anti-HIV [5–7] and DNA topoisomerase activity [8]. In the early 1990s, TCS was applied in the treatment of patients with AIDS or AIDS-related complex in phases I and II studies [9–12]. Abbreviations: TCS, trichosanthin; RIP, ribosome-inactivating protein; FITC, fluorescein isothiocyanate; DMPC, 1,2-dimyristoyl-snglycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol ∗ Corresponding author. Tel.: +86-10-62772214; fax: +86-10-62784768. E-mail address: [email protected] (S.-F. Sui). 0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2003.12.006

Trichosanthin belongs to the type I ribosome-inactivating protein (RIP) family and can inactivate eukaryotic ribosome by removing the A4324 of the 28s rRNA [13,14]. The three-dimensional structure of TCS has been resolved to a resolution of 1.73 and 2.6 Å by two groups [15,16]. And both the genomic and cDNA of TCS have been cloned [17,18]. The membrane/protein interaction plays an important role in RIP’s physiological effect, because these toxins must first be translocated across the cellular membrane before they can inactivate the ribosome in the cytosol. Our previous studies have shown that TCS can spontaneously insert into negatively charged phospholipid membrane under low pH condition [19], similar with the well-studied diphtheria toxin [20,21]. This phenomenon is possibly correlated with its membrane translocation. But the mechanism of the protein/phospholipid interaction remains unclear. The present work aims at the further understanding of the TCS membrane insertion process. In particular, we investigated the interaction force between TCS and phospholipid, and the distribution of TCS in phospholipid monolayer after mem-

106

X.-F. Xia et al. / Colloids and Surfaces B: Biointerfaces 39 (2004) 105–112

brane insertion. These findings provide further evidence to understand the molecular mechanism on TCS membrane insertion under low pH condition.

2. Materials and methods 2.1. Materials FITC, DMPC, DMPG, DPPC and DPPG were purchased from Sigma (St. Louis, MO, USA). The lipid dye probe (rhodamine-DOPE) was purchased from Avanti Polar Lipids (Alabaster, AL). The other chemicals used were of analytical grade and made in China. The deionized water used in the experiment had a resistivity of no less than 18.0 M cm. For experiments under different pH, 50 mM Tris–HCl buffer was used for pH 7.4, while 50 mM sodium acetate–acetic acid (NaAc–HAc) buffer was used for pH 4.6. All the solutions were freshly prepared. 2.2. Purification and fluorescent labeling of TCS TCS was extracted from the root of T. kirilowii (Tianhuafen) according to Zhang et al. [22] with slight modification: The dried slice of Tianhuafen obtained from a local drugstore was homogenized with 50 mM Tris–HCl buffer at pH 6.8 (buffer A) using a high speed blender. The outcome was centrifuged to remove the insoluble. Ammonium sulphate was added to 40% saturation to the supernatant, and the mixture was left for 12 h and centrifuged. The collected supernatant was adjusted to 75% saturation with ammonium sulphate, left for 6 h, and centrifuged. The precipitate was resuspended with buffer A, and dialyzed overnight against buffer A. The resulting solution was applied to a CM-sepharose C-50 column, washed with buffer A, and eluted with buffer A containing 0.3 M NaCl. The elution peak was collected, and put onto the second column of Sephadex G-75, which was previously equilibrated with Buffer A, and eluted under the same conditions. TCS appeared in the second elution peak. Purity determination of TCS showed a single band at the 27 kDa position on SDS-PAGE (silver stain). TCS was FITC labeled following the method described by Nairn [23]. The unlabled FITC and the labeled protein was separated with G-15 gel filtration chromatography. The ratio of fluorochrome to TCS was determined to be about 1:5. 2.3. Measurement of protein adsorption In order to measure the enrichment of TCS beneath the phospholipid monolayer, a monolayer collection method was used, which is based upon monolayer aspiration and fluorescence measurement [24]. The procedure is briefly described as follows. Twenty nannomole of phospholipid (DPPC or DPPG) was spread on the indicated subphase,

and was left 30 min for the solvent to evaporate. Then the monolayer was compressed and kept at a surface pressure of 20 mN/m. FITC labeled TCS solution was then injected into the subphase to a final concentration of 200 nM. The system was left for 5 h until the adsorption process reached equilibrium, and then the subphase surface was carefully collected by vacuum aspiration and directed into a test tube. The concentration of FITC-TCS in this collected solution was determined by its fluorescence intensity. Calibration curves were obtained from the fluorescence intensity of known concentration FITC-TCS in the same buffer as the subphase. The volume of the collected solution is vc , and the protein concentration in vc is cc (with corresponding fluorescence emission intensity θ c ). The protein concentration in the bulk is cb (with corresponding fluorescence intensity θ b ). Here, θc = kc. The surface pressure was kept constant at 20 mN/m, so the surface area was reduced by Ar after monolayer collection. The proteins in the collected solution can be divided into two parts according to where they come from: those from the interface and those from the adsorbed bulk solution. Thus, we can have: vc cc = ΓAr + vc cb Here, Γ is the surface excess of protein caused by protein adsorption onto the phospholipid monolayer. Γ =

1 vc vc (vc cc − vc cb ) = (cc − cb ) = (θc − θb ) Ar Ar Ar k

All parameters on the right side of the above equation can be determined by measurements. The fluorescence intensity measurement was carried out on a HITACHI M850 Fluorescence Spectrophotometer using a 1 cm2 quartz fluorescence cuvette. The emission and excitation slit widths were both set at 5 nm. The excitation wavelength was set at 495 nm and the emission wavelength 520 nm. 2.4. Measurement of the π–A isotherms A KSV5000 LB trough (KSV Instruments Ltd., Finland) was used to obtain the surface pressure–molecular area (π–A) isotherms of mixed protein/phospholipid and pure phospholipid monolayers at the air/water interface. The Teflon trough being 270 mm long and 75 mm wide was placed on an isolated vibration-free table and was ensconced in a glass chamber to avoid contaminants from the environment. Temperature regulation of the trough was controlled by circulating constant-temperature water from an external circulator through the tubes attached to the aluminum-based plate of the trough. The trough was thoroughly cleaned before experiment until the measured surface pressure was below 0.1 mN/m upon complete compression for pure water. During the experiment, 180 ml of indicated solution was added into the trough. Then phospholipid dissolved in chloroform/methanol (3:1, v/v) was

X.-F. Xia et al. / Colloids and Surfaces B: Biointerfaces 39 (2004) 105–112

spread on the surface of the subphase, and left for 0.5–1 h until the organic solvent evaporated. π–A isotherms was then obtained by compressing the protein layer at the air/water interface with two Teflon barriers at a speed of 1 Å2 /(molecule min) at (25 ± 0.5) ◦ C. Each experiment was carefully repeated with good reproducibility, deviation within ±5%. 2.5. Microfluorescence film balance The microfluorescence film balance system was constructed using a Nikon DIAPHOT-TMD inverted epifluorescence microscope and a computer controlled film balance. The Teflon trough of the film balance with dimensions 20 cm × 8 cm × 0.8 cm contains a glass window at the bottom. Monolayers were observed through the glass window with an extremely long working distance objective. The magnification of the objective used for observation was 40×. When the FITC labeled protein was added, the monolayer was observed alternately through two interchangeable cut-off filters, the permissible wavelengths of which corresponded respectively to signals from the rhodamine labeled lipid and FITC labeled TCS. The pictures were received with a low-light-level SIT camera (Hamamatsu c2400) and recorded with a VHS video recorder (Panasonic HD-100). The monolayers were spread from a lipid solution in chloroform/methanol (3:1, v/v) containing 0.2% of the dye probe rhodamine-DOPE.

3. Results 3.1. Adsorption of TCS by negatively charged phospholipid monolayer When FITC-TCS is injected into the subphase under phospholipid monolayer, FITC-TCS will be enriched beneath the monolayer if there is any attractive force between protein and phospholipid. Thus, the fluorescence intensity of the collected surface solution should be stronger than that of the bulk. The adsorption experiments were carried out for two different phospholipids: negatively charged DPPG and neutral DPPC. The surface excess of FITC-TCS were calculated according to the equation given in materials and methods, results listed in Table 1. For negatively charged DPPG monolayer, experiments were carried out under pH 4.6 and 7.4 separately. From the results shown in Table 1, we can see that under both pH conditions, the surface excess of protein is positive, thus TCS was enriched on the surface. Since TCS is a basic protein with isoelectric point (pI) 9.4, the enrichment caused by negatively charged phospholipid might be a result from the electrostatic attraction. This was further confirmed by the experiment carried out for DPPC under pH 4.6, we can see that under this condition the surface excess of FITC-TCS was negligible because DPPC is a neutral phospholipid.

107

The results of Table 1 show also that the enrichment of TCS by DPPG monolayer is stronger under pH 4.6 than 7.4. Comparing with the bulk concentration the surface concentration of TCS has a 30% increase under pH 7.4 while a 50% increase under pH 4.6. This is possibly caused by two reasons: the first is that the protein maybe further positively charged under lower pH, and the increase in surface enrichment is a result from the stronger coulombic attraction. The second reason may come from our previous result [19] that TCS can insert into DPPG monolayer under pH 4.6 while not under pH 7.4, so the excess enrichment may be caused by the TCS molecules inserting in the phospholipid monolayer. 3.2. Incorporation of TCS in phospholipid monolayer by hydrophobic interaction Phospholipid monolayers incorporated with TCS were obtained following the procedure described by Signor et al. [25]: phospholipid was spread on the subphase containing 20 nM TCS. Then the system was left for 3 h for TCS molecules to adsorb onto the air/water interface. Because TCS is a surface-active protein which can spontaneously adsorb to the air/water interface, the initial surface pressure had a 5 mN/m increase due to the protein adsorption. Thus, the result monolayer would be consisted of phospholipid mixed with TCS molecules. The π–A isotherms of different mixed TCS/phospholipid monolayers are shown in Fig. 1. The results showed that once TCS has been incorporated into the either DPPG or DPPC monolayers, it could not be completely squeezed out by compression until the monolayers collapsed. Since this phenomenon is irrelative with the pH condition and the kind of phospholipid used, it could not be caused by the interactions between TCS and the phospholipid headgroup. So, most likely it is the hydrophobic interaction that help to stabilize TCS incorporated in the phospholipid monolayer. In the previous works on bombolitin III [25] or lipid transfer protein [26], it is found that the proteins’ incorporation into phospholipid monolayer is reversible, since the compression isotherms tend to converge with that of pure phospholipid monolayer before the collapse pressure. But here the π–A isotherms for mixed TCS/phospholipid monolayers did not intersect with the π–A isotherms of pure phospholipid monolayers until the collapse pressure. The irreversible Table 1 Adsorption of TCS by phospholipid monolayer Surface excess (ng/mm2 ) DPPG, pH 4.6 DPPG, pH 7.4 DPPC, pH 4.6

2.31 ± 0.25 1.42 ± 0.22 0.06 ± 0.01

The samples of bulk and surface solutions were collected 5 h after FITC-TCS was injected beneath the phospholipid monolayer. The surface pressure was kept constant at 20 mN/m. Surface excess of protein was calculated according to the equation given in Section 2.

X.-F. Xia et al. / Colloids and Surfaces B: Biointerfaces 39 (2004) 105–112

60.00

60.00 Surface pressure (mN/ m)

Surface pressure (mN/m)

108

40.00

20.00

0.00

0.00

(A)

40.00 80.00 Molecular area (Å2/molecule)

120.00

0.0 0

0.00

40.00

80.00

120. 00

40.00

80.00

120. 00

Molecular area (Å2/molecule)

60.00 Surface pressure (mN/ m)

Surface pressure (mN/ m)

20.00

(B)

60.00

40.00

20.00

0.0 0

0.00 (C)

40.00

40.00

80. 00

Molecular area (Å2/molecule)

120.00

40.00

20.00

0.0 0

0.00 (D)

Molecular area (Å2/molecule)

Fig. 1. π–A isotherms of mixed TCS/phospholipid monolayers. The dashed curves were the control isotherms of the pure phospholipid. Mixed monolayers were obtained by spreading phospholipid dissolved in chloroform/methanol (3:1, v/v) on the subphase containing 20 nM TCS. (A) TCS/DPPC monolayer, pH 7.4; (B) TCS/DPPC monolayer, pH 4.6; (C) TCS/DPPG monolayer, pH 7.4; (D) TCS/DPPG monolayer, pH 4.6.

characteristic of TCS incorporation was possibly caused by interaction between TCS and the phospholipid hydrophobic chain. The hydrophobic interaction is strong enough to help TCS to stay stably inside when incorporated in the monolayer. 3.3. Effect of TCS on the microstructure of phospholipid monolayer The effect of TCS on the microstructure of the interfacial film was obtained from the fluorescence micrographs to gather further information on the interaction mechanism. As pointed out above, TCS can insert into DPPG monolayer under pH 4.6. So the influence of TCS on DPPG monolayer under pH 4.6 was investigated in detail by this technique. DPPG containing fluorescent probe rhodamine-DOPE was spread on the surface of pH 4.6, 50 mM NaAc-HAc buffer, and compressed to 14 mN/m after solvent evaporated. Dark domains which have been described as condensed lipid domains coexisting with the fluid phase [27–29] appeared as uniform circular shape under this condition (shown in Fig. 2A and B ). Then TCS was injected into the subphase until a final concentration of 100 nM and the surface pressure began to increase as a result of TCS insertion. The microstructure of the film was examined by fluorescence

microscope at different surface pressures. The results are shown in Fig. 2A and B. These fluorescence micrographs exhibit that TCS insertion causes drastic change in the fluorescence image pattern. At beginning the coalescence between circular dark domains was observed. Then the circular dark domains formed a shape like string of beads (Fig. 2A) and finally became elongate (Fig. 2B). As a control, the fluorescence micrographs for DPPG under pH 7.4 and DPPC under pH 4.6 were also examined after TCS was injected into the subphase. In these cases, the surface pressure did not change indicating there was no TCS inserted into the phospholipid monolayer. Also the domain pattern changed little as shown in Fig. 3. Combination of the results of Figs. 2 and 3 indicates that the shape change of the dark domains mentioned above is due to membrane insertion of TCS, while the adsorption of TCS beneath the monolayer has little influence. Fig. 4A shows the distribution of TCS in the DPPG monolayer (surface pressure 15 mN/m) under pH 4.6 by observing the FITC-labeled TCS with fluorescence microscope. From the image of Fig. 4A, we can see that the emission intensity of the FITC-labeled TCS almost distributed homogeneously in the observation field, no dark region was observed on the monolayer. On the contrary, for the same sample the emission intensity from the dye-lipid of the DPPG monolayer

X.-F. Xia et al. / Colloids and Surfaces B: Biointerfaces 39 (2004) 105–112

109

Fig. 2. Real time epifluorescent observation of DPPG monolayer after TCS insertion. The bulk pH was 4.6. The π–t curve at the bottom showed TCS insertion caused surface pressure increasing. (A) and (B) are fluorescence images of the mixed monolayer taken at indicated times. (A ) and (B ) were the fluorescence images for pure DPPG monolayer under the same surface pressures as (A) and (B). Scale bar shown in all images: 50 ␮m.

displayed a pattern with dark domains (Fig. 4B). This result indicated that TCS could insert into both the condensed and fluid phase of the phospholipid monolayer. If we kept the surface pressure constant at 15 mN/m after TCS was injected beneath the DPPG monolayer under pH 4.6, 8 h later, the monolayer became “homogenized” (Fig. 5). The dark domain was destroyed and the contrast between dark and bright phase became very weak, the image exhibited a gray, unclear appearance.

4. Discussion 4.1. The driving forces dominating membrane insertion of TCS In this work, we investigated the function of electrostatic attraction and hydrophobic interaction in the membrane insertion process of TCS. Adsorption of TCS by phospholipid monolayer showed strong attraction between TCS and

110

X.-F. Xia et al. / Colloids and Surfaces B: Biointerfaces 39 (2004) 105–112

Fig. 3. Fluorescence micrographs for DPPG under pH 7.4 and DPPC under pH 4.6. The monolayer surface pressures were all kept at 15 mN/m. (A) DPPG monolayer before TCS was added into the subphase; (B) DPPG monolayer after TCS was added into the subphase; (C) DPPC monolayer before TCS was added into the subphase; (D) DPPC monolayer after TCS was added into the subphase. Scale bar shown in all images: 50 ␮m.

negatively charged DPPG, both under pH 4.6 and 7.4. But the insertion of TCS into DPPG monolayer only happened under acidic environment. For neutral DPPC monolayer, neither adsorption nor insertion of TCS was observed under any pH condition. We concluded that the electrostatic attraction between TCS and DPPG was essential but not sufficient for TCS insertion.

The hydrophobic interaction between TCS and phospholipid was also examined in the present work. The results showed that when TCS was integrated into the lipid (DPPG or DPPC) monolayer, it could not be completely squeezed out until the monolayer collapsed, and this phenomenon was independent on the kind of phospholipid headgroup, demonstrating a strong hydrophobic interaction between protein

Fig. 4. Distribution of TCS in the DPPG monolayer under pH 4.6. Fluorescence micrographs were imaged from the optical emission of FITC (A) or from the optical emission of rhodamine (B). (A) is the FITC-TCS fluorescence image, showed the distribution of TCS in the monolayer. (B) is the corresponding fluorescence image of DPPG mixed with Rh-DOPE, showed the domain pattern. Scale bar shown in all images: 50 ␮m.

X.-F. Xia et al. / Colloids and Surfaces B: Biointerfaces 39 (2004) 105–112

Fig. 5. Fluorescence micrographs for DPPG 8 h after TCS was added beneath the monolayer. The bulk pH was 4.6. Scale bar: 50 ␮m.

and phospholipid. But since TCS can only insert into DPPG monolayer under low pH condition, conclusion was drawn that although the TCS penetration into membrane was not initiated by hydrophobic interaction, it helped to stabilize the incorporated TCS in the membrane, lead to an irreversible membrane insertion process. It is known that TCS is a water soluble protein, and from its three-dimensional structure we can see that its surface is mostly hydrophilic. It is likely the protein underwent a conformational change after incorporated into the lipid membrane so that the strong hydrophobic interaction is between the exposed hydrophobic residue and the lipid hydrophobic tails. But direct evidence is needed to confirm this assumption. In all, the membrane insertion of TCS may be a combinative effect of electrostatic attraction, hydrophobic interaction and conformational change of TCS [30].

111

monolayer in both liquid and solid phase regions provided further evidence that the hydrophobic interaction between TCS and phospholipid is very strong. After TCS penetrated into the DPPG monolayer under pH 4.6, the shape of the solid domain was changed from circular to elongate. For pure DPPG monolayer, the solid phase exhibits a uniformly circular shape. This is because the attraction interior the orderly packed solid phase is stronger than the attraction between solid and liquid phase. So, the solid phase adopt a circular shape to make maximum molecule staying interior the solid phase. The elongated forms of the dark domains caused by TCS was similar to that observed by Worthman et al. [38] in their study on mixed cholesterol/phosphatidylcholine monolayer. To interpret the elongate domain caused by cholesterol, Johann et al. [39] pointed out that this phenomenon must be a consequence of either a variation in the intermolecular interactions or a manipulation of the energetic situation at the fluid-condensed interface by the reacting molecules adsorbed to the domain boundary. In their work, cholesterol was assumed to preferentially adsorb to the two-dimensional fluid-condensed interface and to reduce the line tension, behaving as a “line-active” agent. But in the present work, since the distribution of TCS in the monolayer was homogeneous, the elongation of domain shape was most likely caused by TCS penetration into the condensed phase and broke the ordered parking of the phospholipid molecules. The destroying of condensed phase by TCS insertion was obvious after 8 h, when the fluorescence image became blur and the difference between the condensed and the fluid phase became unconspicuous. The breakage of ordered phospholipid packing caused by TCS insertion may hinder the hydrophobic interaction among phospholipid tails, which may be the answer for the homogenizing effect.

4.2. Effect of TCS on the microstructure of the phospholipid monolayer Acknowledgements Epifluorescence microscopy was used to study the influence of TCS on the monolayer microstructure. Epifluorescence microscopy of lipid–protein monolayers is a convenient tool for studying protein distribution and lipid– protein interactions at the air/water interface [31–34]. In the present work, fluorescence micrograph showed that TCS inserted into both the fluid and condensed phase of the phospholipid monolayer. It almost distributed homogeneously in the monolayer. The same phenomenon was also observed by Subirade et al. [26], in their work on wheat lipid transfer protein. While for many other proteins, like phospholipase A2 , cytochromes b, pulmonary surfactant proteins B and C, they preferentially partition into the fluid membrane phases [35–37]. The difference may be caused by the strength of the protein–phospholipid interaction. When it is strong enough to break the condensed packing of phospholipid in the solid phase, protein will insert into both the solid and liquid phase of the monolayer, otherwise it may only insert into the liquid part. The fact that TCS can insert into DPPG

This work was supported by the National Natural Science Foundation of China (NSFC).

References [1] J.M. Maraganore, M. Joseph, M.C. Bailey, J. Biol. Chem. 262 (1987) 11628–11633. [2] Y. Wang, R.Q. Qiang, Z.W. Gu, S.W. Jin, L.Q. Zhang, Z.X. Xia, G.Y. Tian, C.Z. Ni, Pure Appl. Chem. 58 (1986) 789–798. [3] K.F. Cheng, Obstet. Gynecol. 59 (1982) 494–498. [4] Y. Chan, M.K. Tong, M.J. Lau, Shanghai J. Chin. Med. 3 (1982) 30–31 (in Chinese). [5] E. De Clercq, Med. Res. Rev. 20 (2000) 323–349. [6] M.S. McGrath, M.S. Hwang, S.E. Caldwell, I. Gaston, K.C. Luk, P. Ng, Y.L. Wu, V.L. Crowe, S. Daniels, J. Marsh, J. Deinhart, T. Leakas, P.V. Vennari, J.C. Yeung, H.W. Lifson, J. D. Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 2844–2848. [7] J. Zhao, L.H. Ben, Y.L. Wu, W. Hu, K. Ling, S.M. Xin, H.L. Nie, L. Ma, G.J. Pei, Exp. Med. 190 (1999) 101–111.

112

X.-F. Xia et al. / Colloids and Surfaces B: Biointerfaces 39 (2004) 105–112

[8] Z.H. Qi, X.S. Jiang, S.D. Liu, X.R. Yang, H.Y. Li, FASEB J. 14 (2000) A1440. [9] V.S. Byers, A.S. Levin, L.A. Waites, B.A. Starrett, R.A. Mayer, J.A. Clegg, M.R. Price, R.A. Robins, M. Delaney, R.W. Baldwin, AIDS 4 (1990) 1189–1196. [10] V.S. Byers, A.S. Levin, A. Malvino, L. Waites, R.A. Robins, R.W. Baldwin, AIDS Res. Humm. Retroviruses 10 (1994) 413–420. [11] J.O. Kahn, L.D. Kaplan, J.G. Gambertoglio, D. Bredesen, C.J. Arri, L. Turin, T. Kibort, R.L. Williams, J.D. Lifson, P.A. Volberding, AIDS 4 (1990) 1197–1204. [12] R.A. Mayer, P.A. Sergios, K. Coonan, L. O’Brien, Eur. J. Clin. Invest. 22 (1992) 113–122. [13] M.X. Li, H.W. Yeung, L.P. Pan, S.I. Chan, Nucleic Acids Res. 19 (1991) 6309–6312. [14] J.S. Zhang, W.Y. Liu, Nucleic Acids Res. 20 (1992) 1271–1275. [15] K.Z. Pan, Y.J. Lin, K.J. Zhou, Z.J. Fu, M.H. Chen, D.R. Huang, D.H. Huang, Sci. China (Seires B) 36 (1993) 1069–1081. [16] J.P. Xiong, Z.X. Xia, Y. Wang, Nat. Struct. Biol. 1 (1994) 695– 700. [17] T.P. Chow, R.A. Feldman, M. Lovett, M. Piatak, J. Biol. Chem. 265 (1990) 8670–8674. [18] P.C. Shaw, M.H. Yung, R.H. Zhu, W.K.K. Ho, T.B. Ng, H.W. Yeung, Gene 97 (1991) 267–272. [19] X.F. Xia, S.X. Wang, J.B. Luo, R.N.S. Wong, S.F. Sui, Chin. Sci. Bull. 44 (1999) 1892–1895. [20] C.R. Alving, B.H. Iglewski, K.A. Urban, J. Moss, R.L. Richards, J.C. Sadoff, Proc. Natl. Acad. Sci. U.S.A. 77 (1980) 1986– 1990. [21] M.G. Blewitt, L.A. Chung, E. London, Biochemistry 24 (1985) 5458–5464.

[22] Y. Zhang, W.L. Li, Q. Hao, G. Yu, Q.S. Li, Q.Z. Yao, Biochem. Biophys. Res. Commun. 197 (1993) 407–414. [23] R.C. Nairn, Fluorescent Protein Tracing, fourth ed., Churchill Livingstone, London, 1976 (Appendix A: 1–5). [24] F. Wang, X.F. Xia, S.F. Sui, Biophys. J. 83 (2002) 985–993. [25] G. Signor, S. Mammi, E. Peggion, H. Ringsdorf, A. Wagenknecht, Biochemistry 33 (1994) 6659–6670. [26] M. Subirade, C. Salesse, D. Marion, M. Pézolet, Biophys. J. 69 (1995) 974–988. [27] M. Losche, C. Helm, H.D. Mattes, H. Mohwald, Thin solid films 133 (1985) 51–64. [28] R.M. Weis, H.M. McConnell, Nature 310 (1984) 47–49. [29] R.M. Weis, Chem. Phys. Lipids 57 (1991) 227–239. [30] X.F. Xia, S.F. Sui, Biochem. J. 349 (2000) 835–841. [31] M. Ahlers, R. Blankenburf, H. Haas, D. Möbius, H. Möhwald, W. Müller, H. Ringsdorf, H.U. Siegmund, Adv. Mater. 3 (1991) 39–46. [32] M.M. Lipp, K.Y.C. Lee, J.A. Zasadzinski, A.J. Waring, Science 273 (1996) 1196–1199. [33] H. Möhwald, Annu. Rev. Phys. Chem. 41 (1990) 441–476. [34] K.J. Stine, Microsc. Res. Technol. 27 (1994) 439–450. [35] D.W. Grainger, A. Reichert, H. Ringsdorf, C. Salesse, FEBS Lett. 252 (1989) 73–82. [36] W.M. Heckl, B.N. Zaba, H. Möhawld, Biochim. Biophys. Acta 903 (1987) 166–176. [37] K. Nag, S.G. Taneva, J. Perez-Gil, A. Cruz, K.M.W. Keough, Biophys. J. 72 (1997) 2638–2650. [38] L.D. Worthman, K. Nag, P.J. Davis, K.M.W. Keough, Biophys. J. 72 (1997) 2569–2580. [39] R. Johann, C. Symietz, D. Vollhardt, G. Brezesinski, H. Möhwald, J. Phys. Chem. 104 (2000) 8512–8517.