KR20140119995A

KR20140119995A - Nanofibers with multiple alpha-helices based on hairpin-type amphiphilic peptides and the Method for preparation thereof

Info

Publication number: KR20140119995A
Application number: KR1020130035285A
Authority: KR
Inventors: 임용범; 한소희
Original assignee: 연세대학교 산학협력단
Priority date: 2013-04-01
Filing date: 2013-04-01
Publication date: 2014-10-13
Also published as: KR101596346B1

Abstract

The present invention relates to nanofibers with multiple alpha-helices based on hairpin-type amphiphilic peptides, wherein the hairpin-type amphiphilic peptide includes a hydrophilic region having an alpha-helix structure and a hydrophobic region. The nanofibers with multiple alpha-helices according to the present invention is expected to be utilized in medical fields, such as biosensor materials and therapeutic agents having bioactivity, since the energy is reduced due to the self-assembly through intermolecular attraction when the nanofibers are prepared, the preparation process is environmentally friendly, the strength of the non-covalent bond can be controlled through the control of the ionic strength, and the thermal stability and the alpha-helix stability are excellent.

Description

[0001] The present invention relates to multiple alpha-helical nanofibers including hairpin-like amphiphilic peptides and methods for preparing the same. [0002] Nanofibers with multiple alpha-helices based on hairpin-type amphiphilic peptides,

The present invention relates to nanofibers comprising multiple alpha-helices comprising an amphipathic peptide in the form of a hairpin having a stabilized alpha-helix, and more particularly to a nanofiber comprising an alpha-helix forming fragment, an arginine rich motif Oligaminization domain of the amphipathic peptide of the present invention is a self-assembled, multi-alpha-helical nanostructure.

Α-helix, the secondary structure of proteins, is the most common structural motif found in proteins and plays a key role in various biological recognition processes both inside and outside the cell, such as protein-DNA, protein-RNA and protein-protein interactions And these interactions are very important in maintaining and controlling the various physiological functions of cells and organisms.

As a result, studies have been actively made to develop a drug capable of controlling or inhibiting various interactions of living organisms by making alpha-helical peptides that are easy to handle and have excellent cell permeability rather than using large proteins as they are.

Therefore, it is preferable to use a self-assembly method for preparing the α-helical peptide. However, the self-assembly method for making a desired form by itself is a simple method in which peptides and nanostructures, which were difficult to manufacture by a conventional top-down approach, It is possible to manufacture by an experimental method, and it has advantages of being biocompatible and environmentally friendly.

The main interactions contributing to this self-assembly are as follows. Typically, hydrogen bonding, which is a weak bonding force between molecules with relatively strong electronegativity and molecules with hydrogen, and nonpolar molecules present in aqueous systems, (Hydrophobic interaction), as well as van der waals interaction and ionic interaction exist in addition to the hydrophobic interaction. These noncovalent interactions exhibit a weak bonding force when forming a single aggregate, but a strong bonding force when forming multiple bonds.

Peptide-based self-assembled nanostructures have excellent molecular recognition and functional flexibility for specific molecules and are expected to be applied in a wide variety of applications compared to conventional organic synthetic material-based nanomaterials. However, when the peptide is not present in the protein state due to the characteristics of the peptide, the secondary structure is easily released and is thermally unstable and vulnerable to environmental changes.

In order to solve the above problems, most of the peptide-based self-assembling nanomaterials developed to date are limited to peptides having a cyclic structure. For example, the patent 10-2010-0075201 discloses a peptide- Forming fragment, which exhibits thermal stability. However, there still exists a limitation that it is based on a cyclic peptide.

As a result, in order to solve the problems of the prior art, the present invention has developed nanofibers composed of a linear peptide having stability of a secondary structure of a peptide and an amphipathic peptide for utilizing peptide nanostructures in various fields.

Accordingly, the present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to provide a method of preparing a peptide having a stable alpha-helix structure, Nanofiber < / RTI >

Another object of the present invention is to provide a method for producing the nanofiber.

Accordingly, in order to achieve the above object, the present invention provides a multiple alpha -helical nanofiber using an amphipathic peptide in the form of a hairpin comprising a hydrophilic region having an alpha -helical structure and a hydrophobic region.

In addition, the hydrophilic region includes an arginine rich motif (ARM) domain forming an alpha -helical structure and a proline rich loop, and the hydrophobic region may include an oligomerization domain.

In addition, the amphipathic peptide may further comprise a hydrophobic functional group.

In addition, the amphipathic peptide may include the amino acid sequences shown below.

[SEQ ID NO: 1] (Rev ₃₄ _-54 )

TRQARRNRRRRWRERQRQIHS

[SEQ ID NO: 2] (Rev ₂₁ _-54 ):

FLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHS

[SEQ ID NO: 3] (Rev ₁₂ _-64 ):

LLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILSTYLK

Also, the hydrophobic functional group may be a saturated fatty acid, and the saturated fatty acid may be selected from the group consisting of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid and docosano And an acid.

In addition, the hydrophobic functional group may be an alkyl group having 1 to 36 carbon atoms.

Also, the multiple alpha-helical nanofibers according to the present invention can be made of V-form dimers including the amphipathic peptides.

Also, the multiple alpha -helical nanofibers according to the present invention can be prepared by self-assembly.

In order to achieve the above object, according to the present invention,

I) preparing an amphipathic peptide,

Ii) removing the terminal protecting group of the amphipathic peptide to form a hairpin structure,

Iii) joining the amphiphilic peptide to obtain a V-form dimer,

And iv) reacting the dimer to produce nanofibers.

The method may further comprise the step of binding a hydrophobic functional group to the hairpin-like amphipathic peptide formed in step ii).

The present invention is based on the formation of self-assembled hairpin peptide monomers through non-covalent bonding of hydrophobic interactions of amphiphilic linear peptides and the formation of nanofibers by noncovalent bonding of the hydrophobic regions of the monomers, Nanofibers are energy-saving and self-assembled by self-assembly through the intermolecular attraction during manufacturing, and can control the strength of non-covalent bonds by controlling the ionic strength and have excellent thermal stability and alpha-helix stability. It is expected to be used in medical fields such as therapeutic agents with biological activity.

FIG. 1 is a conceptual view illustrating the structure of multiple alpha -helical nanofibers using an amphipathic peptide in the form of a hairpin according to an embodiment of the present invention,
(a) represents the structure of the Rev protein of HIV-1,
(b) and (c) are conceptual diagrams showing structures of hairpin-type amphipathic peptides,
(d) is a conceptual diagram showing the structure of multiple alpha -helical nanofibers.
2 is a conceptual diagram showing the structures of Comparative Example 2 (a) and Comparative Example 3 (b) according to the present invention.
3 is a conceptual diagram showing the structures of Production Example 1 (a) and Production Example 2 (b) according to the present invention.
Figure 4 is a CD spectrum measured to confirm the alpha -helical structure of the peptide according to the present invention,
(a) is a CD spectrum obtained by dissolving a peptide according to Comparative Example 1 in water at 4 ° C (dotted line) and dissolving it in PBS (a line)
(b) is a CD spectrum obtained by dissolving the peptides according to Comparative Example 2 in water at 4 캜 (dotted line) and dissolving them in PBS (line)
(c) is a CD spectrum obtained by dissolving the peptides according to Comparative Example 3 in water at 4 ° C (dotted line) and dissolving them in PBS (string)
(d) is a CD spectrum obtained by dissolving the peptide of Comparative Example 3 in PBS at 4 캜 in PBS (line) and 25 캜 in PBS (dotted line).
5 is a CD spectrum measured for comparison between the stabilization of the helix structure of Comparative Example 3 (○) and Preparation Example 1 (●) according to the present invention.
FIG. 6 is a CD spectrum in which peptides according to Production Example 1 of the present invention are dissolved in water and green (blue) in a 50% TFE solvent.
7 is an FP spectrum obtained by dissolving a peptide according to Preparation Example 1 of the present invention in water and a (geen) 50% TFE solvent.
8 is an FP spectrum in which the peptide according to Production Example 1 of the present invention is dissolved in water and λ _max is measured.
9 is an FP spectrum measured by dissolving a peptide according to Preparation Example 1 of the present invention in water,
(a) is the FP spectrum measured at various peptide concentrations, and (b) is the normalized FP spectrum.
10 is a graph showing the solubility of the peptide according to Production Example 1 of the present invention in accordance with ionic strength.
11 is a graph illustrating the measurement of nanofibers containing the peptide according to Production Example 1 of the present invention,
(a) is the CD spectrum measured to compare the stabilization of the alpha-helix structure in pure water or 60 mM KF,
(b) is the CD spectrum measured by dissolving in 60 mM KF at different temperatures,
(c) and (d) are graphs showing the forward scan (orange) and the reverse scan (black) according to the temperature.
12 is a TEM image of nanofibers according to the present invention (Bar = 0.1 mm)
13 is an AFM image of nanofibers according to the present invention.
14 is a MS spectrum of a peptide according to the present invention measured by MALDI-TOF,
(a) is the MS spectrum in which the peptide according to Comparative Example 1 was measured, and
(b) is the MS spectrum of the peptide according to Comparative Example 2,
(c) is the MS spectrum in which the peptide according to Comparative Example 3 is measured, and
(d) is the MS spectrum of Peptide according to Preparation Example 1,
(e) is the MS spectrum in which the peptide according to Production Example 2 was measured.

Hereinafter, the present invention will be described in more detail.

The present invention relates to multiple alpha-helical nanofibers and methods of preparation using amphipathic peptides in the form of hair fins having excellent solubility, thermal stability and structural stability.

The present invention relates to multiple alpha -helical nanofibers using an amphipathic peptide in the form of a hairpin comprising a hydrophobic region and a hydrophilic region, wherein the nanofibers stabilize the alpha -helical structure and simultaneously form a plurality of alpha -helical nanofibers .

The amphipathic peptide is composed of a hydrophilic region and a hydrophobic region and is characterized in that the hydrophobic interaction of the oligomerization domain of the hydrophobic region forms a hairpin structure.

In addition, the amphipathic peptide is a peptide comprising an amino acid sequence that forms an alpha -helical structure, and is preferably an amphipathic peptide comprising a hydrophilic region and a hydrophobic region, more preferably an alpha-helical peptide, A peptide including an arginine rich motif (ARM) domain forming a helix structure, and may include any one selected from the group consisting of the following Structural Formulas 1 to 3:

In addition, the hydrophilic region of the amphipathic peptide includes an arginine-rich motif (ARM) domain of alpha-helical structure consisting of 17 amino acids, and may further include a proline-rich loop.

In addition, the hydrophobic region of the amphipathic peptide includes an oligomerization domain and can form a hairpin structure by hydrophobic interaction between the amino acid molecules of the oligomerization domain.

The amphiphilic peptide may further include a hydrophobic functional group at the N-terminus and the C-terminus. The hydrophobic functional group may be an alkyl group having 1 to 36 carbon atoms, preferably stearic acid, , [Structural formula 5].

The amphipathic peptide must contain either a hydrophobic domain in the peptide or a hydrophobic functional group for the formation of nanofibers.

Previous researches have been conducted on macrocyclic structures, in which the molecules are constrained in the cyclic structure, the alpha-helix structure is primarily stabilized, and the self-assembling peptide moiety becomes more rigid when manufactured into a nanostructure, And is stabilized secondarily. However, such conventional techniques are limited to cyclic peptides that do not contain linear peptides, and are formed by cyclization of macromolecules through covalent bonds, which is a disadvantage in that it is difficult to manufacture.

In the meantime, the present invention relates to a method for producing a nanostructure using a linear peptide and self-assembling the nanostructure by a hydrophobic action, which is a strong non-covalent bond between N-terminal and C-terminal, stabilizing the alpha -helical structure in the nanofiber, cyclization effect ", that is, a pseudo-cyclization effect, which is easier to manufacture than the prior art, is environment-friendly, and has excellent thermal and structural stability.

Through this, it is possible to replace the alpha -helical structure stabilized through the non-covalent bonding of the nanostructure formed of the conventional cyclic compound with the nanostructure of the present invention.

[Structural formula 1] Rev ₃₄ _-54 peptide

TRQARRNRRRRWRERQRQIHS

[Structural formula 2] Rev ₂₁ _-54 peptide

FLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHS

[Structural Formula 3] Rev ₁₂ _-64 peptide

LLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILSTYLK

[Structural Formula 4] SA-Rev ₁₂ _-64 peptide amphiphile

Stearate-LLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILSTYLK

[Structural Formula 5] SA-Rev ₁₂ _-64 -SA peptide amphiphile

Stearate-LLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQI HSISERILSTYLK-Stearate

Also, the nanofibers formed by self-assembly are characterized by having a thickness of 14 nm. After formation of a V-shaped dimer through tail-to-tail noncovalent bonding in the hairpin structure of the Rev amphipathic peptide, The two hydrophobic heads of the dimer are self-assembled by oligomerization to form nanofibers. The hydrophobic portion of the dimer is directed toward the inside of the nanofiber, and the structure of the peptide and the nanofiber in more detail is shown in FIGS. 1, 2, and 3.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram showing a structure of a hairpin-like amphipathic peptide and nanofibers according to an embodiment of the present invention. FIG. FIG. 1A is a conceptual diagram illustrating a domain structure of Rev peptide according to another embodiment of the present invention. FIG. 1B is a SA-Rev ₁₂ _-64 or SA-Rev ₁₂ _-64 -SA according to another embodiment of the present invention. schematic diagram showing the structure of an amphipathic peptide, and Figure 1c is an alpha of SA-Rev ₁₂ _-64 in accordance with yet another embodiment of the present invention is a conceptual view showing a model of a stabilized hairpin structure including a helix, can also 1d FIG. 6 is a conceptual diagram showing dimers and nanofibers formed by self-assembly of Rev ₁₂ _-64 peptide according to another embodiment of the present invention. FIG.

The present invention is based on a hydrophilic arginine domain and promotes self-assembly by hydrophobic interaction of an oligomerization domain to obtain a hairpin-type monomer, and through the tail-to-tail noncovalent bonding of the alkyl group with the domain of the hydrophobic domain Nanofibers are formed, and alpha-helix is stabilized through self-assembly without chemical modification.

Another aspect of the present invention provides a method for producing multiple alpha -helical nanofibers using an amphipathic peptide in the form of a hair pin, which comprises the following steps.

I) preparing an amphipathic peptide,

Iii) joining the amphipathic peptide produced in the above step to obtain a V-form dimer,

And iv) reacting the dimer to produce nanofibers.

Hereinafter, the present invention will be described in detail with reference to the following Production Examples and Experimental Examples. It will be apparent to those skilled in the art that the following Preparation Examples and Experimental Examples are merely illustrative of the present invention and that the present invention is not limited by the following Production Examples and Experimental Examples.

Comparative Examples 1 and 2: Preparation of Rev ₃₄ _-54 Peptide and Rev ₂₁ _-54 Peptide

The peptide was preferentially synthesized from Fmoc-Ser-OH, which is the first amino acid of the peptide sequence of the present invention, in 2-chlorotritylazine, which is a solid phase. The amino acid synthesis after the synthesis of Fmoc-Ser-OH was synthesized using a Tribute peptide synthesizer from Protein technologies inc. The standard amino acid protecting group was used for the synthesis.

To isolate the peptide from the resin, the peptide bound to the resin was treated with a separation solution (trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water = 95: 2.5: 2.5) for 3 hours Next, the mixture was pulverized using tert-butyl methyl ether. The resulting peptide was purified by reverse phase HPLC (water-acetonitrile, 0.1% TFA).

Molecular weight was determined by MALDI-TOF mass spectrometry. The concentration of peptides synthesized using molar extinction coefficients of tyrosine (1280 M-1 cm-1) and tryptophan (5690 M-1 cm-1) in water / acetonitrile (1: Respectively.

Comparative Example 3. Rev ₁₂ _-64 peptide

Peptides were synthesized on synthetic Rink amide MBHA resin LL (novabiochem) using Tribute peptide synthesizer (protein technologies, inc) according to standard protocols. To protect the Lys side chain at the C-terminus of the peptide, an acid-labile methoxytrityl (Mmt) was coupled to the ortho position using a protecting group.

Production Example 1. SA-Rev ₁₂ _-64 Amphipathic peptide

Except that the peptide prepared according to Comparative Example 3 was further immobilized with a hydrophobic functional group.

SA-Rev ₁₂ _-64, ethanol (TFE) / AcOH Mmt the functional groups of the Lys side chain with methylene chloride (MC) / 2,2,2- trifluoro to synthesize the amphiphilic peptides (6: 2: 2) To remove the protecting group. HBTU and DIPEA were used to bond stearic acid to the ε-amine group of the Lys residue.

Production Example 2. SA-Rev ₁₂ _-64 -SA Amphipathic peptide

SA-Rev ₁₂ _-64 -SA In order to synthesize an amphipathic peptide, steric acid was simultaneously bound to the N-terminal α-amine group of the Lys side chain and the ε-amine group to the C-terminal.

Experimental Example 1. Circular dichroism analysis

In order to confirm the distribution of the alpha -helical structure in the peptides of Comparative Examples 1 to 3 prepared according to the present invention, a circular dichroism spectroscopic analysis was performed and proceed as follows.

The CD spectra were measured using a Chirascan Circular Dichroism spectrometer equipped with a Peltier temperature controller. Spectra were recorded between 260 nm and 190 nm using a cuvette with a path of 2 mm or 10 mm. The scan was repeated five times to obtain an average. It was also calculated by molar ellipticity per amino acid residue. Peptide concentrations were typically 15 μM, unless otherwise noted, and sample solutions were incubated for at least two days prior to measurement. The results are shown in FIG. 4, the dotted line being dissolved in water, and the normal line being dissolved in PBS. The samples used in Experimental Example 1 were carried out at 4 ° C.

4A, peptides prepared according to Comparative Example 1 were each dissolved in water and dissolved in PBS (15 mM potassium phosphate, 150 mM potassium fluoride, pH 7.4) to analyze the circular dichroism spectroscopy. In Comparative Example 1, the Rev ₃₄ _-54 peptide consisting of 21 amino acids contained an alpha-helical form of the ARM domain and a portion of the oligomerization domain at the C-terminus. As a result of the spectroscopic spectrum, it was confirmed that the peptide had a definite minimum ellipticity of 200-203 nm . It is present in the form of random coils and does not have a secondary structure of alpha-helix.

In Comparative Example 2, the Rev ₂₁ _-54 peptide comprising a part of the oligomerization domain in the ARM (Arginine rich motif) domain, and N- and C- terminal ends of the each of PBS (15 mM potassium phosphate, 150 mM potassium fluoride, pH 7.4) and pure water. As a result, the ellipticity of α-Helix of 222 nm was confirmed. In order to compare the results of CD analysis, the ellipticity at 208 nm vs. the ellipticity at 222 nm The ratio was calculated as [?] ₂₂₂ / [?] ₂₀₈ . [θ] ₂₂₂ / [θ] ₂₀₈ is sensitive to the dihedral angles of the back bone and can be used to indicate the degree of stabilization of the alpha-helix. When dissolved in PBS, the ratio of [θ] ₂₂₂ / [θ] ₂₀₈ was 0.48, which was twice as high as that of 0.28 dissolved in pure water. This shows that the increase in ionic strength in PBS enhances the hydrophobic interactions and increases the secondary structure helicity.

FIG. 4C is a Rev ₁₂ _-64 peptide according to Comparative Example 3, wherein a complete oligomerization domain is bonded to the N-terminal and C-terminal to form a hairpin structure, and an amino acid And the ratio of [[theta]] ₂₂₂ / [theta] ₂₀₈ was 0.28 in pure water and 0.48 in PBS, and the stability of the helix structure similar to those of Comparative Examples 1 and 2 was shown, 1 and random coils of the same type exist.

The oligomerization domain that forms the hairpin structure, but without the additional interaction for the stabilization of the helix structure, such as protein-RNA, the helix structure, which is the secondary structure, exists in a relaxed state.

4D is a CD spectrum obtained by measuring the temperature of a sample obtained by dissolving the peptide of Comparative Example 3 in PBS at 4 DEG C and 25 DEG C, respectively.

As shown in Figure 4 (d), the stability of the alpha-helical structure of the peptide decreases with increasing temperature. As a result, it was confirmed that the alpha -helix structure of the peptide had the secondary structure of the peptide released at 25 DEG C due to thermal instability.

The above results show that the stabilization degree of the helix structure of Comparative Example 1 which does not form a hairpin shape and that of Comparative Example 3 which is a hairpin type are similar to each other. When dissolved in PBS, the helix structure is partially relaxed But it was insignificant and negligible, and it was confirmed that the degree of stabilization decreased slightly with temperature change. Considering that the human body temperature is generally 37 ° C, it can be seen that other changes are needed to stabilize the helix structure.

Thus, the addition of an alkyl chain to peptides according to Comparative Examples 1 to 3 of the present invention enhanced hydrophobic interactions to stabilize alpha-helix in amphipathic peptides and nanofibers.

Experimental Example 2

The peptides prepared according to Comparative Example 3 were dissolved in water (100 μM) and the peptide prepared according to Preparation Example 1 was dissolved in water (100 μM) to measure CD spectra. The procedure was the same as in Experimental Example 1 above.

As shown in FIG. 5, the degree of stabilization of the helix structure according to the solvents of Comparative Example 3 and Production Example 1 was compared. In Fig. 5, ◯ represents an aqueous solution (100 μM) of the peptide prepared according to the above Comparative Example 3, and  represents an aqueous solution (100 μM) of the peptide prepared according to Preparation Example 1.

The ratio of [?] ₂₂₂ / [?] ₂₀₈ increased from 0.29 to 0.39 when the amphiphilic peptide according to Comparative Example 3 was further added with an alkyl group having a hydrophobic functional group in comparison with the water-soluble amphipathic peptide (100 μM) Could know. As a result, it was confirmed that the alpha -helix structure is more stably retained when the hydrophobic functional group is further included than the existing amphiphilic peptide.

Experimental Example 3.

The SA-Rev ₁₂ _-64 amphipathic peptide according to Preparation Example 1 was dissolved in water (0.6 [mu] M) and 50% 2,2,2-trifluoroethanol, a helix stabilizer that stabilizes the formation of the alpha -helical structure (0.6 μM) was dissolved in TFE to measure the circular dichroism, and the stability of the α-helical structure in the peptide according to the solvent was compared. The CD measurement process of this experiment was performed in the same manner as in Experimental Example 1.

As a result, as shown in FIG. 6, the amphipathic peptide solution using TFE as a co-solvent had a high [[theta]] ₂₂₂ / [theta] ₂₀₈ ratio of 0.76 as expected, and the amphiphilic peptide solution Which is about three times higher than that of the control group. That is, it was confirmed that the α-helix structure was more stably retained when dissolved in TFE than in water or PBS.

Experimental Example 4.

(0.6 μM) solution prepared by dissolving SA-Rev ₁₂ _-64 amphipathic peptide prepared in Preparation Example 1 in water (0.6 μM) dissolved in 50% 2,2,2-trifluoroethanol (TFE) Fluorescence polarization spectroscopy (FP) was used to measure the solution, and the measurement method is described in more detail below.

Fluorescence spectra were measured using a Chirascan spectrometer equipped with FP.3 fluorescence polarization accessory (Applied photohysics). The spectrometer was calibrated as a well known anisotropy sample and subjected to general fluorescence staining (Pose Bengal staining). The spectra were measured in the range of 300 nm to 280 nm with a 10 mm path-length cuvette using a 320 nm cutoff filter. The scans were measured 5 times and the average was recorded.

As a result, as shown in FIG. 7, a peak excitation wavelenght ( _max ) at 286-287 nm was observed as a positive band, indicating that when the band is sensitive to Brownian molecular motion and contains tryptophan in alpha-helix, . When dissolved in TFE rather than dissolved in water, the λ _max was changed from 287 nm to 286.4 nm, a more acute form.

8 is a graph showing the results of fluorescence spectroscopic analysis of a solution of SA-Rev ₁₂ _-64 amphipathic peptide prepared in Preparation Example 1 dissolved in water at a concentration ranging from 0 to 100 μM. According to FIG. 8, as the peptide concentration initially increased,? _Max gradually decreased and then suddenly decreased rapidly. It is believed that the stability of the helix structure is limited by the Brownian molecular motion, and discontinuous λ _max changes are believed to occur when the peptide concentration reaches some point.

Experimental Example 5

In order to measure the solubility of the amphipathic peptide according to the ionic strength, the SA-Rev ₁₂ _-64 amphipathic peptide according to Preparation Example 1 was dissolved in various concentrations of KF solution to measure the FP.

The SA-Rev ₁₂ _-64 amphipathic peptide according to Preparation Example 1 was dissolved in 15 μM of each KF solution in a concentration range of 0-150 mM. After incubation overnight with ultrasound, the supernatant was centrifuged at maximum speed (RCF: 16110 x g) for 5 minutes, and the absorbance of the supernatant was measured with a NanoDrop spectrophotometer (Thermo Scientific). The results are shown in Figures 9 and 10 using an average value measured three times. As a result, as shown in FIG. 10, it was confirmed that as the ionic strength was increased, the peptide gradually decreased.

Experimental Example 6.

The nanofibers prepared using the SA-Rev ₁₂ _-64 amphipathic peptide according to Preparation Example 1 were dissolved in water (15 μM peptide) and the other was dissolved in 60 mM KF (15 μM peptide) , And CD spectra were measured at 25 캜 to compare the stability of the alpha -helical structure in the nanofibers.

Since Cl ions have strong UV absorbance at low wavelengths, KF (potassioum fluoride) is generally used because it is preferred to use saturated NaCl to increase the ionic strength when analyzing CD spectra. Based on the results of Experimental Example 5 And 60 mM KF solution, respectively.

As a result, as shown in Fig. 11A, 0.35 in the case of dissolving in water, 0.65 in case of dissolving in 60 mM KF solution, and about 2 times higher in case of dissolving in 60 mM KF solution, and the change with temperature in Fig. .

However, as shown in FIG. 11C, the ratio of [?] ₂₂₂ / [?] _{208 was} also found to increase with increasing temperature as the temperature of the sample was measured to be 0.74 at a high temperature of 74 ° C, It is believed to have secured nanofibers containing a superior helix structure. The increased hydrophobic interactions at elevated temperatures show that the extreme thermal stability of Sa-Rev ₁₂ _-64 , coupled with hydrophobic functional groups, and the alpha-helix structure are exhibited by the hairpin structure.

Also, referring to FIG. 11D, it can be seen that the occurrence of hysteresis during the forward and reverse scan is related to the kinetic effect of self-assembly. It was confirmed that the ellipticity decreased at 222 nm, but the decrease of [θ] ₂₂₂ / [θ] ₂₀₈ was insignificant.

Experimental Example 7.

The multiple alpha -helical nanofibers prepared using the amphipathic peptide according to Preparation Example 1 were measured by transmission electron microscopy (TEM).

Transmission electron microscopy is carried out by placing a 10 μm sample (typically 15-100 μM) on a carbon-coated copper grid, incubating for 1 minute, removing the excess solution of the sample with filter paper, Negative stain with 1% uranyl acetate. Observations were made using JEOL-JEM 2010 at 120 kV and the data were analyzed using Digital Micrograph software and the results are shown in FIG.

Experimental Example 8.

The multiple alpha-helix nanofibers prepared using the amphipathic peptide according to Preparation Example 1 were measured by an atomic force microscope (AFM), the atomic force microscope scans were scanned at 1.2 to 1.5 V at 0.5 Hz, The results are shown in Fig.

As shown in FIG. 12 and FIG. 13, the multiple alpha -helical nanofiber according to the present invention was in the form of nanofibers which are usually shown, and the thickness of the nanofibers was 14 nm.

Experimental Example 9

Molecular weights were measured by MALDI-TOF mass spectrometry of Comparative Examples 1 to 3 and Preparation Examples 1 and 2. The concentration of peptides synthesized using molar extinction coefficients of tyrosine (1280 M-1 cm-1) and tryptophan (5690 M-1 cm-1) in water / acetonitrile (1: Respectively. The results are shown in Fig.

Claims

A multiple alpha-helix nanofiber comprising an amphipathic peptide in the form of a hairpin comprising a hydrophilic region having an alpha-helix structure and a hydrophobic region.

The method according to claim 1,
Wherein the hydrophilic region comprises an arginine rich motif (ARM) domain forming an alpha-helical structure and a proline rich loop,
Wherein the hydrophobic region comprises an oligomerization domain. &Lt; RTI ID = 0.0 > 8. < / RTI >

The method according to claim 1,
Wherein said amphiphilic peptide further comprises a hydrophobic functional group.

The method according to claim 1,
Wherein the amphiphilic peptide comprises an amino acid sequence of [SEQ ID NO: 1] to [SEQ ID NO: 3] below.
[SEQ ID NO. 1] (Rev ₃₄ _-50 )
TRQARRNRRRRWRERQRQIHS
[SEQ ID NO. 2] (Rev ₂₁ _-54 )
FLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHS
[SEQ ID NO: 3] (Rev ₁₂ _-64 )
LLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISE RILSTYLK

The method of claim 3,
Wherein the hydrophobic functional group is a saturated fatty acid and wherein the saturated fatty acid is selected from the group consisting of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid and docosano &Lt; RTI ID = 0.0 > helix < / RTI > nanofibers.

The method of claim 3,
Wherein the hydrophobic functional group is an alkyl group having 1 to 36 carbon atoms.

The method according to claim 1,
Wherein the nanofiber comprises a V-form dimer comprising the amphipathic peptide. &Lt; RTI ID = 0.0 > 8. < / RTI >

The method according to claim 1,
Wherein the nanofibers are formed by self-assembly. &Lt; RTI ID = 0.0 > 8. < / RTI >

A method for producing multiple alpha-helical nanofibers using hairpin-like amphiphilic peptides,
I) preparing an amphipathic peptide;
Ii) removing the terminal protecting group of the amphipathic peptide to form a hairpin structure;
Iii) joining the amphipathic peptide produced in the above step to obtain a V-form dimer; And
And iv) reacting the dimer to produce a nanofiber. The method for producing multiple alpha-helix nanofibers using the amphipathic peptide.

10. The method of claim 9,
Further comprising the step of binding a hydrophobic functional group to the hairpin-like amphipathic peptide formed in step ii).