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
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Abstract |
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Keywords: design/fibrils/peptides/physico-chemical characteristics
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Introduction |
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This result opens new possibilities for the application of 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
FFP does not change the ability of the peptide to form coiled-coil fibrils (Potekhin et al., 2001
). 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., 1997b
). 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.
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Materials and methods |
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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 045% 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, 1983), as well as by staining with amido black (Schaffner and Weissmann, 1973
) and with G-250 (Bradford, 1976
).
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 0.10.5 mg/ml. The molar ellipticity [] was calculated from the equation:
where []obs is the ellipticity measured in degrees at the wavelength
, 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
-helicity has been calculated as described in Chen et al. (1974
).
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, 1971) 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. (1990). The laser power was 100200 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, 1983). 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., 1968). Carbon films of 0.2 nm on freshly cleaved mica were prepared using an electron beam evaporator (Vasiliev and Koteliansky, 1979
). 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., 2000). The peptide concentrations ranged from 1.0 to 1.2 mg/ml. The data were analyzed after scan rate normalization and baseline subtraction. The vant Hoff enthalpy was calculated using the usual relationship (Privalov and Potekhin, 1986
):
where 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.
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Results |
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The original FFP forms fibrils at acid pH and spherical aggregates at neutral pH (Potekhin et al., 2001
). The transition between the two states is absolutely reversible, highly cooperative and occurs at pH 5.56.0. At this pH, glutamic acid is the most probable group to be deprotonated. However, the shifting of glutamic acid pKa 4.24.4 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., 1999
; Suzuki et al., 1999
). Therefore, it was suggested that the electrostatic repulsion between glutamate residues at positions (g) of the
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
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: FFP-1, QLARQL(QQLARQL)4 and
FFP-2, QLARSL(QQLARSL)4. It was also decided to synthesize a peptide
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 FFP-1, -2 and -3 studied at room temperature. In contrast to the original
FFP (Potekhin et al., 2001
), the new peptides have a high content of
-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
-helical conformation. The ellipticity ratio at 220 and 208 nm is
0.961.01, which was suggested to be typical of interacting
-helices (Zhou et al., 1994
). As seen from the figure, the
-conformation of
FFP-1 (Figure 1c) and
FFP-3 (Figure 1d) is not changed at least up to pH 11.0. The
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
-helical one, with slight turbidity of the solution. The insert in Figure 1b shows the titration curve of peptide
FFP-2; it is seen that transition from the
-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
FFP-2 and
FFP (Potekhin et al., 2001
) differ greatly in the half-width of transition.
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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 2.53.0 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 FFP-2 peptides in alkaline conditions leads to the formation of shapeless aggregated material.
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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 FFP-3. Figure 3 shows thick bundles of
FFP-3 formed after binding together of at least five protofilaments.
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To assess the dimensions of fibrils in solution, sedimentation and diffusion of the preparations were performed. Figure 4 shows sedimentation profiles for FFP-2 and
FFP-1 at acid pH. Like the original
FFP,
FFP-2 is represented as a single narrow symmetrical peak at 6.42S, which is close to the sedimentation coefficient of the initial
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, 1971
).
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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 Svedbergs equation. Using the data given in the table, we have calculated Perren form factors (Cantor and Schimmel, 1980) for the studied preparations. They are 6.01, 4.72 and 7.97 for peptides
FFP,
FFP-2 and
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
FFP and
FFP-1, and 150 for
FFP-2. Thus,
FFP-2 is able to form markedly longer fibrils than the other two. Unfortunately, such measurements for
FFP-3 could not be done owing to a low solubility of the preparation.
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We have found that fibrils formed from FFP-1,
FFP-2 or
FFP-3 peptides are extremely stable. Similar to the original
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., 2001
), 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, 1996
), 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 23 K) (Table II) and the wide minor peak in the pre-denaturation range (290330 K) decreases or disappears. At the same time, the enthalpy of the basic transition increases.
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Discussion |
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The peptide modification does affect the fibril stability, though not in a crucial way. Both FFP-1 and
FFP-2 have a highly positive net charge in contrast to neutral
FFP and
FFP-3. The electrostatic repulsion of positive charges can explain the decrease in the stability of
FFP-1 and
FFP-2 fibrils when compared with the original
FFP or
FFP-3. The stability of
FFP-3 fibrils is higher than that of
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
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 FFP-1 and
FFP-3 have no conformational changes at least up to pH 11. Inasmuch as
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,
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 FFP-2. As seen from titration curves of
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
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 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 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
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., 1997b
).
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Acknowledgements |
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Received July 7, 2003; revised October 27, 2003; accepted October 30, 2003