Shift of fibril-forming ability of the designed {alpha}-helical coiled-coil peptides into the physiological pH region

T.N. Melnik1, V. Villard2, V. Vasiliev1, G. Corradin2, A.V. Kajava3 and S.A. Potekhin1,4

1Institute of Protein Research, Russian Academy of Sciences,142290 Pushchino, Moscow Region, Russia, 2Institute of Biochemistry, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland and 3Centre de Recherches de Biochimie Macromoléculaire, CNRS FRE-2593, 1919 Route de Mende, 34293 Montpellier, Cedex 5, France

4 To whom correspondence should be addressed. e-mail: spot{at}vega.protres.ru


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently, we designed a short {alpha}-helical fibril-forming peptide ({alpha}FFP) that can form {alpha}-helical nanofibrils at acid pH. The non-physiological conditions of the fibril formation hamper biomedical application of {alpha}FFP. It was hypothesized that electrostatic repulsion between glutamic acid residues present at positions (g) of the {alpha}FFP coiled-coil sequence prevent the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers axial growth of the fibril. To test this hypothesis, we synthesized {alpha}FFPs where all glutamic acid residues were substituted by glutamines or serines. The electron microscopy study confirmed that the modified {alpha}FFPs form nanofibrils in a wider range of pH (2.5–11). Circular dichroism spectroscopy, sedimentation, diffusion and differential scanning calorimetry showed that the fibrils are {alpha}-helical and have elongated and highly stable cooperative tertiary structures. This work leads to a better understanding of interactions that control the fibrillogenesis of the {alpha}FFPs and opens opportunities for their biomedical application.

Keywords: design/fibrils/peptides/physico-chemical characteristics


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The {alpha}-helical coiled-coil structures are recognized as one of nature’s favorite ways of creating an oligomerization motif. Amino acid sequences of the known coiled-coil structures have a characteristic heptad repeat, (abcdefg)n, with apolar residues in positions (a) and (d) and polar residues generally elsewhere. Proteins with the coiled-coil pattern form two-, three-, four- and five-stranded oligomers depending on variations in their sequences (O’Shea et al., 1991Go; Harbury et al., 1993Go, 1994; Malashkevich et al., 1996Go). The rules governing the stoichiometry of coiled-coils are still intensively tested. We focused our research on the five-stranded coiled-coils (Kajava, 1996Go; Terskikh et al., 1997aGo). The analysis of their structures suggested that (i) widening of the hydrophobic surface of the {alpha}-helix containing apolar (a) and (d) residues by addition of apolar residues at positions (e) or (g) as well as (ii) certain interhelical ionic and hydrogen bonds between side chains in the (b), (c), (e), (f) and (g) positions cause the formation of the five-stranded {alpha}-helical coiled-coils (Kajava, 1996Go). The design of coiled-coils with a priori predicted properties is a convincing way to prove our understanding of the principles of coiled-coil oligomerization. Recently, we designed a peptide that was predicted to form a five-stranded coiled-coil fibril with {alpha}-helices staggered along the axis. The experiments confirmed that the peptides [called {alpha}-helical fibril-forming peptides ({alpha}FFPs)] spontaneously form {alpha}-helical fibrils with a 2.5 nm diameter at acid pH (Potekhin et al., 2001Go). Such a diameter of the fibrils, the {alpha}-helical conformation of the peptide and the orientation of {alpha}-helices along the fibril axis agree well with the anticipated five-stranded arrangement. A more comprehensive electron microscopy study suggested that, in addition to the five-stranded protofilaments, the fibrils have another predominant state corresponding to the dimer of the protofilaments (Kajava et al., 2003Go). In recent years the field of the molecular design of {alpha}-helical fiber structures has grown (reviewed in Yeates and Padilla, 2002Go), but, to our knowledge, this is the first {alpha}-helical peptide able to form soluble nanofibrils of such small diameter. Previous attempts to obtain {alpha}-helical coiled-coil peptides with a potential for axial growth led to fibrous structures, which have a large thickness ranging from ~40 to ~70 nm (Pandya et al., 2000Go; Ogihara et al., 2001Go; Ryadnov and Woolfson, 2003Go).

This result opens new possibilities for the application of {alpha}FFP in biotechnology and medicine. In particular, it was demonstrated that an addition of five to eight amino acid residues to the N-terminus of {alpha}FFP does not change the ability of the peptide to form coiled-coil fibrils (Potekhin et al., 2001Go). When designing such a peptide, we had in mind the practical goal of creating a valuable scaffold for the construction of multivalent fusion proteins; in particular, the coiled-coil with the highest number of subunits is especially promising for this purpose (Terskikh et al., 1997bGo). The integration of approximately 100 biologically active peptides in a single fibrillar structure would significantly enhance the efficiency of their binding due to the multivalency of the complex formed. However, the fibrils were formed only at acid pH, and this property limited the number of potential medical applications. Therefore, it was interesting to modify the designed coiled-coil peptide so that it could self-assemble into fibrils at physiological pH.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peptide synthesis

Peptides were synthesized on a peptide synthesizer (Applied Biosystems 431A) and purified by RP-HPLC (SS 250x1 cm Nucleosil 300-7 C18 column) using a 0–45% CH3CN gradient in 0.1% TFA/H2O for 30 min with a flow rate of 3 ml/min. The purity of the peptides was analyzed by RP-HPLC (C18 analytical column) and mass spectrometry. Peptide concentrations were determined by the method of Waddell based on the difference between spectrophotometric absorptions at 215 and 225 nm (Wolf, 1983Go), as well as by staining with amido black (Schaffner and Weissmann, 1973Go) and with G-250 (Bradford, 1976Go).

Circular dichroism (CD) measurements

CD spectra were obtained on a JASCO-600 spectropolarimeter (Japan Spectroscopic Co.) equipped with a temperature-controlled holder in 0.1 mm thick cells at a peptide concentration of 0.1–0.5 mg/ml. The molar ellipticity [{theta}] was calculated from the equation:

where [{theta}]obs is the ellipticity measured in degrees at the wavelength {lambda}, Mres is the mean residue molecular weight of peptide, C is the peptide concentration (g/l) and L is the optical pathlength of the cell (mm). The percentage of {alpha}-helicity has been calculated as described in Chen et al. (1974Go).

Sedimentation experiments

Sedimentation experiments were performed in 0.1 M NaCl, 10 mM sodium phosphate, pH 2.8 buffer solutions using a Beckman Model E analytical ultracentrifuge with the Schlieren optical system. The sedimentation coefficient was evaluated at a speed of 42 040 r.p.m. by a standard procedure (Bowen, 1971Go) at 20°C.

Diffusion experiments

Diffusion coefficients were determined at 20°C from dynamic light scattering experiments using a spectrometer described in detail in Timchenko et al. (1990Go). The laser power was 100–200 µW. Light scattering was measured at an angle of 90°.

An arrival-time correlator was used to extend the range of correlation times. The data were processed using the ‘Incorrectness’ program developed at the Institute of Protein Research, RAS (Danovich and Serdyuk, 1983Go). The program allows one to estimate diffusion coefficients for a mixture of particles from the equation for the normalized correlation first-order function g(1)(t):

where ai is the portion of scattered radiation of the ith component with the translational diffusion coeffcient Di, and q is the scattering vector module.

Electron microscopy

The samples were negatively stained with 1% aqueous uranyl acetate using the single-layer carbon technique (Valentine et al., 1968Go). Carbon films of 0.2 nm on freshly cleaved mica were prepared using an electron beam evaporator (Vasiliev and Koteliansky, 1979Go). Electron micrographs were taken with a JEM-100C electron microscope at an accelerating voltage of 80 kV and magnification of 80 000.

Calorimetric measurements

Calorimetric measurements were made on a precision scanning microcalorimeter SCAL-1 (Scal Co. Ltd, Russia) with 0.33 ml glass cells at a scanning rate of 1 K/min and under a pressure of 2.5 atm (Senin et al., 2000Go). The peptide concentrations ranged from 1.0 to 1.2 mg/ml. The data were analyzed after scan rate normalization and baseline subtraction. The van’t Hoff enthalpy was calculated using the usual relationship (Privalov and Potekhin, 1986Go):

where {Delta}Hcal is the calorimetric enthalpy, Tm is the apparent temperature of denaturation, Cp,max is the excess heat capacity at the trace maximum and R is the gas constant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peptide design

The original {alpha}FFP forms fibrils at acid pH and spherical aggregates at neutral pH (Potekhin et al., 2001Go). The transition between the two states is absolutely reversible, highly cooperative and occurs at pH 5.5–6.0. At this pH, glutamic acid is the most probable group to be deprotonated. However, the shifting of glutamic acid pKa 4.2–4.4 to 5.5 requires a specific surrounding. Indeed, such a protonation is observed for glutamic acid, which is involved in repulsive electrostatic interactions (Dürr et al., 1999Go; Suzuki et al., 1999Go). Therefore, it was suggested that the electrostatic repulsion between glutamate residues at positions (g) of the {alpha}FFP coiled-coil sequence prevents the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers the axial growth of the fibril. To enable {alpha}FFPs to form fibrils at neutral pH, we substituted all glutamic acid residues by hydrophilic but uncharged glutamines or by serines.

Thus, the following peptides were synthesized: {alpha}FFP-1, QLARQL(QQLARQL)4 and {alpha}FFP-2, QLARSL(QQLARSL)4. It was also decided to synthesize a peptide {alpha}FFP-3, QLAQQL(QQLAQQL)4 where all charged groups, arginine and glutamic acid, are substituted for glutamines.

CD spectroscopy

We used CD spectroscopy to determine the conformation of the synthesized peptides. Figure 1 shows the results of pH titration of the new peptides {alpha}FFP-1, -2 and -3 studied at room temperature. In contrast to the original {alpha}FFP (Potekhin et al., 2001Go), the new peptides have a high content of {alpha}-helical conformation (>95%) not only at acid, but also at neutral pH. The spectra have a maximum at 198 nm and two minima at 208 and 222 nm, characteristic of {alpha}-helical conformation. The ellipticity ratio at 220 and 208 nm is ~0.96–1.01, which was suggested to be typical of interacting {alpha}-helices (Zhou et al., 1994Go). As seen from the figure, the {alpha}-conformation of {alpha}FFP-1 (Figure 1c) and {alpha}FFP-3 (Figure 1d) is not changed at least up to pH 11.0. The {alpha}FFP-2 (Figure 1b) has noticeable conformational changes accompanied by alterations in the CD spectrum when pH is above 9.0. Two specific minima in the spectrum disappear, and at alkaline pH the peptide has a conformation differing from the {alpha}-helical one, with slight turbidity of the solution. The insert in Figure 1b shows the titration curve of peptide {alpha}FFP-2; it is seen that transition from the {alpha}-helical to the non-helical conformation occurs in a wide range of pH (from 9.0 to 12.0) and may reflect the titration of arginine or of the terminal NH2 group. The peptide does not acquire the initial conformation for a period of a few days on return from alkaline to neutral pH. Most likely this is a consequence of the slow kinetics of dissociation of aggregates formed in the alkaline region. It should be noticed that the titration curves for {alpha}FFP-2 and {alpha}FFP (Potekhin et al., 2001Go) differ greatly in the half-width of transition.



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Fig. 1. Far-UV CD spectra of: (a) {alpha}FFP at pH 2.7 (), 3.2 (- - -), 4.5 (– · –), 5.4 (–· · –), 6.2 (······) and 8.0 (—); (b) {alpha}FFP-2 at pH 2.9 (—), 3.9 (– – –), 5.5 (), 7.6 (— —), 8.7 (······), 10.5 (–· · –), 11.2 (- - -), 11.7 (– · –) and 12.8 (– – –); (c) {alpha}FFP-1 at pH 2.9 (—), 3.1 (– – –), 4.1 (– – –), 6.0 (–· · –), 7.2 (–· · –), 9.3 (······), 11.0 (- - -) and 12.3 (– · –); (d) {alpha}FFP-3 at pH 2.9 (—), 6.1 (– – –) and 8.5 (······). Spectra were recorded at a peptide concentration of 0.1–0.5 mg/ml in 10 mM sodium phosphate buffer at 20°C. Inserts show pH dependence of [{theta}]220nm values.

 
Electron microscopy

The electron microscopy study of the synthesized peptides shows that all of them are able to form fibrils both at acid and neutral pH. Two types of fibrils are observed: thin fibrils of 2.5–3.0 nm in diameter which are apparently five-stranded protofilaments, and twice thicker fibrils (Figure 2). In this case spherical particles seen earlier on the original peptide at neutral pH are not observed at all. Aggregation of {alpha}FFP-2 peptides in alkaline conditions leads to the formation of shapeless aggregated material.



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Fig. 2. Electron micrographs of {alpha}FFP-2 peptides at pH 2.8 negatively stained by uranyl acetate. Two types of fibrils are seen, their diameters differing approximately two times (marked with single and double arrowheads). The insert shows paracrystalline inclusions observed in the preparations (here {alpha}FFP-1 at the same magnification and pH 7.3). The diameter of single-stranded fibrils estimated from the dimensions of such paracrystals is 27 Å.

 
Close inspection of the images shows that thick fibrils are formed by association of two protofilaments. This is consistent with our previous electron microscopy study of the original {alpha}FFP that revealed single and double protofilaments (Kajava et al., 2003Go). In some cases, the protofilaments are self-oriented in well ordered paracrystalline arrays (inset in Figure 2). Such crystalline formations give an opportunity to measure more accurately the diameter of the protofilament which is 2.7 ± 0.2 nm. The ability of the protofilaments to form paracrystals suggests that they are not flexible, but have an essentially rigid structure.

The observed ratio of single versus double filaments varies significantly. It changes not only from sample to sample but also from field to field of the same sample. Therefore, it does not reflect the real ratio of single and double fibrils in solution. It is possible to assume that double fibrils as well as paracrystals can appear during adsorption of the protofilaments on the surface of a carbon substrate.

It is interesting that a few thicker bundles can be found in the preparations of {alpha}FFP-3. Figure 3 shows thick bundles of {alpha}FFP-3 formed after binding together of at least five protofilaments.



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Fig. 3. Electron micrograph of {alpha}FFP-3 peptides at pH 7.2 negatively stained by uranyl acetate. In addition to usual strands, uncommonly thick bundles can also be seen.

 
Hydrodynamics

To assess the dimensions of fibrils in solution, sedimentation and diffusion of the preparations were performed. Figure 4 shows sedimentation profiles for {alpha}FFP-2 and {alpha}FFP-1 at acid pH. Like the original {alpha}FFP, {alpha}FFP-2 is represented as a single narrow symmetrical peak at 6.42S, which is close to the sedimentation coefficient of the initial {alpha}FFP measured in the same conditions. Although it is evident that the fibril length of the preparation is heterogeneous, this might have no effect on the sedimentation constant and peak diffusion. This is expected since for strongly elongated particles of a constant diameter the sedimentation coefficient depends only a little on their length (Bowen, 1971Go).



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Fig. 4. Sedimentation profiles of peptides {alpha}FFP-2 (a) and {alpha}FFP-1 (b) in 10 mM sodium phosphate buffer, 0.1 M NaCl, pH 2.8. The profile was obtained in 32 min after the beginning of centrifugation. Peptide concentration was 0.7 mg/ml.

 
In contrast to the above preparations, heterogeneity of {alpha}FFP-1 is well seen on its sedimentation curves. As seen from Figure 4, the preparation contains two fractions with different sedimentation constants. Taking into account that sedimentation constants for both fractions are rather high, it is very likely that the fractions have a fibrillar structure. On the other hand, the sedimentation constant for elongated structures is almost proportional to the square of the section diameter. Thus, it can be postulated that {alpha}FFP-1 fibrils, which are present in solution, can have different sections. It is possible that the minor fraction represents two protofilament fibrils detected with electron microscopy.

The results of measuring the sedimentation constants and diffusion coefficients are listed in Table I. The table shows also molecular weights of the preparations calculated by Svedberg’s equation. Using the data given in the table, we have calculated Perren form factors (Cantor and Schimmel, 1980Go) for the studied preparations. They are 6.01, 4.72 and 7.97 for peptides {alpha}FFP, {alpha}FFP-2 and {alpha}FFP-1, respectively. From this analysis it is apparent that all the peptides assemble in rather elongated structures with a large-to-small radius ratio of more than 200 for {alpha}FFP and {alpha}FFP-1, and 150 for {alpha}FFP-2. Thus, {alpha}FFP-2 is able to form markedly longer fibrils than the other two. Unfortunately, such measurements for {alpha}FFP-3 could not be done owing to a low solubility of the preparation.


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Table I. Hydrodynamic parameters of fibrils
 
Calorimetry

We have found that fibrils formed from {alpha}FFP-1, {alpha}FFP-2 or {alpha}FFP-3 peptides are extremely stable. Similar to the original {alpha}FFP, the structures formed by these peptides remain stable in an aqueous solution at least up to 130°C, both at neutral and acid pH. As in our previous paper (Potekhin et al., 2001Go), we used DMSO as a destabilizing agent of peptide structures. Figure 5 shows temperature dependencies of molar heat capacity of the preparations at high DMSO concentrations. Although the fibrils retain an unusually high stability even in DMSO as compared with other proteins (Kovrigin and Potekhin, 1996Go), they are cooperatively melted at a temperature higher than 365 K. As seen from the figure, the curves have heat absorption peaks that might correspond to cooperative disruption of the structure. That the process is reversible is confirmed by repeated heating of the preparations. It is likely that the preliminary heating of the preparations orders and stabilizes the structure of the fibrils. The main heat absorption peak shifts to higher temperatures (by 2–3 K) (Table II) and the wide minor peak in the pre-denaturation range (290–330 K) decreases or disappears. At the same time, the enthalpy of the basic transition increases.



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Fig. 5. Temperature dependencies of partial molar heat capacities of {alpha}FFP, {alpha}FFP-2, {alpha}FFP-1 and {alpha}FFP-3 in 10 mM sodium phosphate buffer, pH 2.5 at 60% of DMSO (a) and in 75% DMSO (b). {alpha}FFP (—), {alpha}FFP-1 (– – –), {alpha}FFP-2 (– · –), {alpha}FFP-3 (······). The bold lines correspond to second heatings of peptides. Peptide concentrations were from 0.9 to 1.2 mg/ml.

 

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Table II. Thermodynamic characteristics of peptide denaturation in DMSO solution
 
The structure of {alpha}FFP-3 has the highest stability. This structure is stable up to 130°C even in 60% DMSO. Its disruption can be observed only in 75% DMSO at 395.5 K. Thus, its stability exceeds that of the original {alpha}FFP by 23.5 K. In contrast to {alpha}FFP-3, peptides {alpha}FFP-2 and {alpha}FFP-1 have a lower stability as compared with that of {alpha}FFP. In 60% DMSO the stability of {alpha}FFP-1 and {alpha}FFP-2 is correspondingly 15 and 26 K lower than that of the original {alpha}FFP.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The main result of our study is that we succeeded in modifying the de novo designed {alpha}FFP (Potekhin et al., 2001Go) in such a manner that it can form fibrils not only at acid pH but at neutral pH as well. The fact that the substitution of glutamic acid residues at position (g) of the {alpha}FFP coiled-coil sequence shifts the fibril-forming ability of the designed peptides into physiological conditions has clearly demonstrated the correctness of our hypothesis about the factors governing the formation of the fibrils. Another important conclusion is that the fibril-forming ability is not a unique feature of the originally synthesized {alpha}FFP peptide (Potekhin et al., 2001Go; Kajava et al., 2003Go) because the modified peptides were able to form morphologically similar fibrils. The exception is {alpha}FFP-3; in addition to single and double protofilaments it also has very thick fibrils. The existence of the thick fibrils can be explained by a better complementarity of the hydrogen bond interactions between the protofilaments provided by the glutamine residues of {alpha}FFP-3 than by charged residues of the other {alpha}FFPs. It is noticeable that the ratio observed for single and double protofilaments varies significantly. This ratio changes not only from sample to sample, but also in different fields of the same sample. Therefore, it is probable that the formation of double protofilaments, at least in some cases, is connected with the preparation of the sample for electron microscopy. On the other hand, a fraction with a larger sedimentation coefficient than that in the main body of the experimental material was found in the {alpha}FFP-1 preparation. It cannot be excluded that this fraction represents the double protofilament fibrils that are seen on electron micrographs.

The peptide modification does affect the fibril stability, though not in a crucial way. Both {alpha}FFP-1 and {alpha}FFP-2 have a highly positive net charge in contrast to neutral {alpha}FFP and {alpha}FFP-3. The electrostatic repulsion of positive charges can explain the decrease in the stability of {alpha}FFP-1 and {alpha}FFP-2 fibrils when compared with the original {alpha}FFP or {alpha}FFP-3. The stability of {alpha}FFP-3 fibrils is higher than that of {alpha}FFP. This result may be considered as a support of our assumption of the electrostatic repulsion between glutamic acid residues presented at positions (g) of the original {alpha}FFP coiled-coil sequence. The substitution of negatively charged glutamic acid for glutamine may eliminate this repulsion and increase the fibril stability.

The titration curves obtained by CD spectroscopy show that the fibrils formed with peptides {alpha}FFP-1 and {alpha}FFP-3 have no conformational changes at least up to pH 11. Inasmuch as {alpha}FFP-3 has no titrated changed side groups, it may be concluded that titration per se of the N-terminal amino group and the C-terminal carboxyl group cannot affect the ability of the peptides to form fibrils. On the other hand, {alpha}FFP-1 contains arginines at position (f) that can also undergo titration, but at very high pH. As expected, titration to pH 11 does not affect the formation of fibrils.

Attention should also be paid to one more feature of titration of the initial peptide and {alpha}FFP-2. As seen from titration curves of {alpha}FFP, fibrils are disrupted at pH ~6.0, probably due to the anomalous titration of glutamic acid residues. The half-width of the transition shows that the process is cooperative. Fibrils of {alpha}FFP-2 are disrupted as a result of titration of arginines, the process being non-cooperative.

It was also shown that the substitution of residues at the coiled-coil position (g) of {alpha}FFP might favor the formation of well ordered paracrystalline arrays. This is an important result not only for an accurate estimation of the diameter of the protofilaments but also for a further application of these peptides in optics and nanotechnology. Indeed, when oriented such nanofibrils can form materials with an anisotropic transmitting capability.

The ability of a series of {alpha}FFPs to form fibrils at physiological conditions also opens new perspectives for their application in biotechnology and medicine. For example, a soluble oligomer with such a large number of subunits is especially promising as a scaffold for the construction of multivalent fusion proteins. In this case, a large number of copies of the biologically active ligands may protrude from the fibril body and impart high multivalency to the complex. This property of {alpha}FFP can be widely used in medical treatments and biotechnological processes where a higher efficiency can be achieved by associating a larger number of functional subunits into one complex (Terskikh et al., 1997bGo).


    Acknowledgements
 
We thank D.Prokhorov for the assistance in the diffusion experiments and V.Brossard for the peptide purification. The authors gratefully acknowledge the financial support from the Swiss National Science Foundation (grant no. 7SUPJ062317) and from the Russian Foundation for Basic Research (grant no. 02-04-48737).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received July 7, 2003; revised October 27, 2003; accepted October 30, 2003





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