CN102911258B - Method for forming two-dimensional polypeptide nanosheets by regulating assembly of polypeptides with organic micromolecules - Google Patents
Method for forming two-dimensional polypeptide nanosheets by regulating assembly of polypeptides with organic micromolecules Download PDFInfo
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- CN102911258B CN102911258B CN201210234629.3A CN201210234629A CN102911258B CN 102911258 B CN102911258 B CN 102911258B CN 201210234629 A CN201210234629 A CN 201210234629A CN 102911258 B CN102911258 B CN 102911258B
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Abstract
The invention relates to a method for forming two-dimensional polypeptide nanosheets by regulating assembly of polypeptides with organic micromolecules, belonging to the technical field of biological materials. The method comprises the following steps: (1) selecting a molecule self-assembly polypeptide, and selecting an organic micromolecule regulator; (2) thoroughly mixing the polypeptide molecule and the regulator molecule to form a mixed solution; and incubating at 37 DEG C for 7 days. The molecule regulator is introduced on the molecular level to regulate the polypeptides to form a two-dimensional self-assembly sheet structure. The scanning tunnel microtechnique on the molecular level observes that the regulator molecule can regulate the surfactant-like polypeptide assembly structure. The invention provides the interaction site between the regulator molecule and polypeptide on the molecular level, so that people can clearly understand the polypeptide assembly mechanism under the regulation of the regulator molecule.
Description
Technical field
The invention belongs to technical field of biological materials, specifically, the present invention relates to a kind of method of introducing the assembling of conditioning agent molecular regulation polypeptide and then forming two-dimentional polypeptide nano thin slice.
Background technology
As everyone knows, molecular self-assembling has become a kind of very effective approach of nanostructure of preparing, and it can be without external interference in the situation that, by intermolecular each species specific interaction, the nanostructure that inducing molecule height of formation is orderly.In biological and relevant with biology field, this technology is widely used especially.Some biomolecules or class biomolecules, for example peptide and protein, they can utilize intermolecular hydrogen bonding to interact, and the self-assemblies such as hydrophobic interaction form one dimension or zero-dimension nano structure, for example 1-dimention nano fiber, nanotube; Zero-dimension nano ball.People can be applied in every field widely by these nanostructures.For example, the fibroplastic scaffolding structure of 1-dimention nano can be applied in organizational project, and zero-dimension nano spherical structure can be applied in the field of drug delivery.But, so far, almost not studies have reported that, utilize the assembling of polypeptide to form the nanostructure of sequential 2 D.Two-dirnentional structure is a kind of very important geometry equally, and it can possess unique surface properties, unique electrical properties, and can be used widely in all many-sides such as the processing preparations of device.Between 2010-2011; professor Zuckermann of Lorentz Berkeley National Laboratory of the U.S. utilizes a kind of class polypeptide polymer of specially sequence; self-assembly has prepared a large amount of ultra-thin two-dimension nanocrystals; this research can help people to understand the mechanism of peptide and protein assembling by a kind of assembling process of non-natural polymeric system; and for people further design preparation two-dimentional protein nano structure established solid basis (can be with reference to M.Reches; E.Gazit; Science 2003; 300,625; E.Gazit, G.Rosenman, Journal of the American Chemical Society 2010,132,15632; R.N.Zuckermann, et al, Nature Materials 2010,9,454; R.N.Zuckermann, Journal of the American Chemical Society 2011,133,20808.).
Summary of the invention
Research based on above-mentioned is known, and preparing a kind of two-dimentional protein nano structure has very important theory significance and practical significance.The present invention has developed a kind of novel method of utilizing organic molecule end regulation and control polypeptide assembling and then forming two-dimentional polypeptide lamellar structure.
The object of the invention is to, a kind of method of preparing polypeptide two-dimensional nanostructure is provided, carry out according to following step:
(1) select a kind of molecular self-assembling polypeptide, and select a kind of small organic molecule regulators,
(2) described peptide molecule is fully mixed with conditioning agent molecule, form mixed solution; Under 37 DEG C of conditions, hatch 7 days.
In wherein said step (1), be a kind tensio-active agent polypeptide for the preparation of the polypeptide of two-dimensional slice structure, concrete structure is NH
2-KKKFAFAFAFAKKK-COOH, NH
2-KKKFFFFFFFFKKK-COOH, NH
2-AAAAAAAAKKK-COOH, NH
2-DDDFFFFFFFFDDD-COOH, NH
2-DDDAAAAAAAAKKK-COOH, NH
2-DDDFAFAFAFADDD-COOH etc., preferably class tensio-active agent peptide molecule is NH
2-KKKFAFAFAFAKKK-COOH.
Conditioning agent molecule described in wherein said step (2) is pyrimidine, pyrazine, imidazoles, pyroles nitrogen heterocyclic molecule and derivative thereof, for example 4 ' 4-dipyridyl, vinylpyridine, and three pyridines, pyrimidine etc., preferred described tagged molecule is 4,4'-Bipyridine.
The mol ratio of wherein said peptide molecule and conditioning agent molecule is 1:1
In sum, the object of the invention is to develop a kind of novel method of preparing two-dimentional polypeptide nano structure, by adding conditioning agent to regulate and control the packaging assembly of polypeptide on molecular level, impel polypeptide to form two-dimentional assembling primitive, finally form complete polypeptide two dimension assembling lamellar structure.
The present invention at least has following beneficial effect:
The present invention utilizes and introduces molecular regulation agent on molecular level, and regulation and control polypeptide has formed a kind of two-dimentional self-assembly lamellar structure.The present invention utilizes technique of scan tunnel microscope on molecular level, to observe the regulation and control of conditioning agent molecule to class tensio-active agent polypeptide packaging assembly.On molecular level, provide conditioning agent molecule and polypeptide interactional site, can make people be well understood to the mechanism of conditioning agent molecular regulation polypeptide assembling.
The present invention utilizes atomic force microscopy and transmission electron microscopy to disclose polypeptide and the assembling of conditioning agent molecule has formed two-dimensional slice structure.Further prove the validity regulating and controlling on molecular level.
The Young's modulus of the two dimension assembling lamellar structure that the present invention utilizes quantitative nano mechanics imaging technique to study to be formed by polypeptide and conditioning agent molecule, has further surveyed the mechanical property of this two-dirnentional structure.New approaches are provided for developing a kind of novel Bio-Nano-Materials.
Brief description of the drawings
Fig. 1 is schematic arrangement and the combination energy of polypeptide in the time that conditioning agent molecule is combined by different way of class tensio-active agent polypeptide and Molecular regulator; Wherein a is the molecule of class tensio-active agent polypeptide and structural representation and the cartoon schematic diagram of conditioning agent molecule 4,4'-Bipyridine molecule, and b, for peptide molecule is in the time that conditioning agent molecule is combined with two kinds of different modes, simplifies cohesive energy calculation value.
Fig. 2 is the scanning tunnel microscope image that peptide molecule and conditioning agent molecule form common packaging assembly, wherein, a is peptide molecule and conditioning agent molecule 4, the scanning tunnel microscope image of the assembly that 4 '-dipyridyl forms, b is the structural representation of the assembly of peptide molecule and the formation of conditioning agent molecule;
Fig. 3 is the atomic force microscope images that peptide molecule and conditioning agent molecule are assembled the aggregate of rear formation altogether;
Fig. 4 is the transmission electron microscope image that peptide molecule and conditioning agent molecule are assembled the aggregate of rear formation altogether;
Fig. 5 is atomic force microscope images and the Young's modulus measurement image of peptide molecule and the conditioning agent molecule aggregate of assembling altogether rear formation; A is the height image that peptide molecule and conditioning agent molecule are assembled the aggregate of rear formation altogether, b is the Young's modulus measurement image that peptide molecule and conditioning agent molecule are assembled the aggregate of rear formation altogether, c is the statistics height measurements that peptide molecule and conditioning agent molecule are assembled the aggregate of rear formation altogether, and d is the statistics Young's modulus value of peptide molecule and the conditioning agent molecule aggregate of assembling altogether rear formation.
Fig. 6 is the research that peptide molecule and conditioning agent molecule are assembled the aggregate mechanical property of rear formation altogether, a is the pattern that peptide molecule and conditioning agent molecule are assembled the aggregate of rear formation altogether, b is after the power of 2nN is sheared, peptide molecule and conditioning agent molecule are assembled the pattern of the aggregate of rear formation altogether, c and d are for before and after shearing, and peptide molecule and conditioning agent molecule are assembled the high resolution pattern of the aggregate of rear formation altogether.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Embodiment 1 is based on Scanning Probe Microscopy and transmission electron microscope is observed respectively class tensio-active agent polypeptide and Molecular regulator is assembled the two-dimensional nano lamellar structure of formation altogether, and utilizes the mechanical property of quantitative nano mechanics imaging system research polypeptide nano thin slice.
1, the chemical structure of the material that uses
Peptide molecule (NH
2-KKKFAFAFAFAKKK-COOH), the chemical structure of 4,4'-Bipyridine quasi-molecule (4Bpy), as follows:
Class tensio-active agent polypeptide
4 ' 4-dipyridyl (4 ' 4-bipyridyl, 4Bpy)
2, concrete grammar
1) design a kind of class tensio-active agent polypeptide that contains 8 hydrophobic groupings, wherein 8 amino acid are respectively FAFAFAFA, 4 phenylalanines (F) and 4 L-Ala (A), in order to regulate the water-soluble of polypeptide, respectively at 3 Methionins of two tip designs (K) of polypeptide, final polypeptide structure is NH2-KKKFAFAFAFAKKK-COOH, and the hydrophobic amino acid in this polypeptide structure will play an important role in polypeptide assembling process, impels polypeptide to form bar Gansu Province structure.In order to form two-dimentional packaging assembly, we introduce a kind of end conditioning agent molecule 4,4, and ' dipyridyl molecule (4Bpy), is connected in each polypeptide end, impels polypeptide to form the laminated structure that two dimension connects.Polypeptide structure and 4Bpy molecule are optimized, utilize molecular simulation to calculate, specifically adopt density functional theory, energy is combined in the effect that calculates respectively 4Bpy molecule and polypeptide end-COO group and-NH2 group.The theory support (as shown in Figure 1) that adjusted agent molecule is combined with polypeptide;
Fig. 1 is schematic arrangement and the combination energy of polypeptide in the time that conditioning agent molecule is combined by different way of class tensio-active agent polypeptide and Molecular regulator; Wherein a is the molecule of class tensio-active agent polypeptide and structural representation and the cartoon schematic diagram of conditioning agent molecule 4,4'-Bipyridine molecule, and b, for peptide molecule is in the time that conditioning agent molecule is combined with two kinds of different modes, simplifies cohesive energy calculation value.
2) first peptide molecule and Pyridine Molecules are mixed in the aqueous solution, ultrasonic 1 minute, after fully mixing, at 37oC temperature, hatch 7 days, after this, take out 15 microlitre solution, drip to the graphite surface of new cleavage, leave standstill 10 minutes, make mixed molecules system on graphite, form assembly deposition from the teeth outwards after, then dry up with high pure nitrogen.
Utilize commercial multiple mode scanning probe microscope (SPM, Nanoscope IIIa type, Veeco company, the U.S.), experiment condition is constant current mode under atmosphere, and polypeptide and 4 ' 4-dipyridyl system are scanned, and obtains STM high resolution image (as shown in Figure 2);
Fig. 2 (a) is peptide molecule and the conditioning agent molecule STM image in HOPG surface-assembled structure.We find, the assembly formation laminated structure of polypeptide/4Bpy, and polypeptide exists with bar Gansu Province structure, and 4Bpy molecule is followed between Gansu Province of polypeptide, interacts with polypeptide.Conditioning agent molecule links together peptide molecule as tackiness agent on two-dimentional yardstick.The model schematic diagram of polypeptide and 4Bpy interaction of molecules is as shown in Fig. 2 (b) below.By the STM test-results obtaining, we can learn, the intermolecular hydrogen bonding that in the COOH group of polypeptide carboxyl terminal and 4Bpy molecule, N atom forms has driven the formation of polypeptide/4Bpy assembly.In test, the polypeptide length that we measure is 4.5nm, considers carboxyl and the amido size of end group, and the basic and theoretical length of length that this polypeptide is measured matches.
3) by peptide molecule and the Pyridine Molecules mixing solutions of hatching after 7 days, take out 15 microlitres, drip to the mica surface of new cleavage, leave standstill 10 minutes, the aggregate of mixed molecules system is fully adsorbed at mica surface, then dries up with high pure nitrogen.
Utilize commercial multiple mode scanning probe microscope (SPM, Nanoscope V-type, Bruker company, the U.S.), experiment condition is to rap pattern under atmosphere, and the aggregate system of polypeptide and 4Bpy formation is scanned, and obtains afm image (as shown in Figure 3);
Fig. 3 is the two-dimensional nano thin slice pattern that peptide molecule and 4Bpy molecular self-assembling form.
4) by peptide molecule and the Pyridine Molecules mixing solutions of hatching after 7 days, take out 2 microlitres, drip on the copper mesh with carbon supporting film, leave standstill 2 minutes, the aggregate of mixed molecules system is fully adsorbed on copper mesh, then remove surplus solution.
The aggregate pattern that utilizes Philips CM20 LaB6 transmission electron microscope observation polypeptide and 4Bpy molecule to form.(as shown in Figure 4);
The transmission electron microscope image that Fig. 4 is peptide molecule and conditioning agent molecule 4Bpy assembles the aggregate of formation altogether.
5) by peptide molecule and the Pyridine Molecules mixing solutions of hatching after 7 days, take out 15 microlitres, drip to the mica surface of new cleavage, leave standstill 10 minutes, the aggregate of mixed molecules system is fully adsorbed at mica surface, then dries up with high pure nitrogen.
Utilize commercial multiple mode scanning probe microscope (SPM, Nanoscope V-type, Bruker company, the U.S.), experiment condition is quantitative nano mechanics imaging pattern under atmosphere, measures the mechanical property of the two-dimensional nano thin slice of polypeptide and the formation of 4Bpy molecule.Specifically as illustrated in Figures 5 and 6;
Fig. 5 (a) is the height image that peptide molecule and conditioning agent molecule 4Bpy assemble the two-dimensional nano thin slice of formation altogether, its altitude distribution is 4nm, concrete as shown in Fig. 5 (c), the height of substrate is 0nm, and the height of the two-dimensional slice that polypeptide assembling forms is obviously different from substrate level.Fig. 5 (b) is the Young's modulus image that peptide molecule and conditioning agent molecule 4Bpy assemble the two-dimensional nano thin slice of formation altogether.By measuring, the Young's modulus that we obtain two-dimensional nano thin slice is 200MPa, and as shown in Fig. 5 (d), this result shows that polypeptide and conditioning agent molecule form a kind of biological nano soft material.By its surperficial Young's modulus measurement is learnt, the Young's modulus on surface is 230MPa, as shown in Fig. 5 (d), this does not conform to the Young's modulus of mica, this result proves, mica surface has adsorbed the assembly structure that one deck polypeptide and 4Bp form, this structure influence the measurement of Young's modulus of mica.
The present invention utilizes technique of scan tunnel microscope and atomic force microscopy and transmission electron microscopy to observe respectively, introduce the packaging assembly of conditioning agent molecular regulation amyloid polypeptide, and utilize quantitative nano mechanics imaging system, the mechanical property of having studied polypeptide assembling thin slice, specifically comprises the steps:
1) prepare peptide molecule solution, ultrasonic 30 seconds;
2) in the solution of peptide molecule, add conditioning agent molecule, it is fully mixed, hatch 7 days;
3) hatch after 7 days, by the solution of polypeptide solution and polypeptide and the blend of conditioning agent molecule, respectively get 15 microlitres and drop in respectively the high oriented graphite surface of cleavage and the mica surface of cleavage, leave standstill 5 minutes;
4) by the solution of polypeptide solution and polypeptide and the blend of conditioning agent molecule, get 2 microlitres and drop on the copper mesh with carbon supporting film, leave standstill 2 minutes;
5) blow away remaining in surperficial solution with high pure nitrogen;
6) observe with scanning tunnel microscope, observe the common packaging assembly of polypeptide and conditioning agent molecule.
7) observe by atomic force microscope the aggregate structure that aforementioned polypeptides and conditioning agent molecule form.
8) aggregate structure forming with transmission electron microscope observation aforementioned polypeptides and conditioning agent molecule.
9) Young's modulus of the aggregate structure forming with quantitative nano mechanics imaging system research polypeptide and conditioning agent molecule, and other mechanical property of this structure.
Fig. 6 (a) is for before atomic force probe shears, the feature image of the two-dimensional slice structure that peptide molecule and 4Bpy molecule form, A-D mark the lamellar structure of formation, selection D is research object, other three thin slices thing that serves as a mark, proves the research of original position.In the time using the power of 2nN to handle nano flake, found that, after scan operation, nano flake is sheared into two sections, as shown in Figure 6 (b).This result proves that the nano flake structure that polypeptide and 4Bpy form is a kind of soft structure, is easily sheared.Fig. 6 (c and d) be the high resolution feature image of nano flake before and after shearing.Image obviously shows, after shearing, lamellar structure dwindles, and proves in operating process, and this kind of soft structure is extremely easily out of shape, and is more prone to be pruned.
By above-mentioned five steps, we prepare and have characterized a kind of two-dimensional nano lamellar structure obtaining of assembling altogether by polypeptide and conditioning agent molecule, and this research provides a novel method for preparing the Bio-Nano-Materials of new function.
Claims (1)
1. prepare a method for polypeptide two-dimensional nanostructure, it is characterized in that carrying out according to following step:
(1) select a kind of molecular self-assembling polypeptide, and select a kind of small organic molecule regulators,
(2) described peptide molecule is fully mixed with conditioning agent molecule, form mixed solution; Under 37 DEG C of conditions, hatch 7 days;
The mol ratio of wherein said peptide molecule and conditioning agent molecule is 1:1;
Wherein the described molecular self-assembling polypeptide of step (1) is NH
2-KKKFAFAFAFAKKK-COOH;
Wherein the described conditioning agent of step (2) is 4 ' 4-dipyridyl.
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| CN101514225A (en) * | 2008-10-13 | 2009-08-26 | 西安蓝晶生物科技有限公司 | Self-polymerization polypeptide and preparation method and application thereof |
| CN101939329A (en) * | 2007-08-30 | 2011-01-05 | 滑铁卢大学 | Amino acid pairing-based self assembling peptides and methods |
| CN102115498A (en) * | 2009-12-31 | 2011-07-06 | 华中科技大学同济医学院附属协和医院 | Connexin core sequence-containing amphiphilic polypeptide and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101939329A (en) * | 2007-08-30 | 2011-01-05 | 滑铁卢大学 | Amino acid pairing-based self assembling peptides and methods |
| CN101514225A (en) * | 2008-10-13 | 2009-08-26 | 西安蓝晶生物科技有限公司 | Self-polymerization polypeptide and preparation method and application thereof |
| CN102115498A (en) * | 2009-12-31 | 2011-07-06 | 华中科技大学同济医学院附属协和医院 | Connexin core sequence-containing amphiphilic polypeptide and application thereof |
Non-Patent Citations (1)
| Title |
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| 许小丁等.多肽分子组装.《中国科学：化学》.2011,第41卷(第2期),第221-228页. * |
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