Micelle-Induced Self-Assembling Protein Nanowires: Versatile Supramolecular Scaffolds for Designing the Light-Harvesting System Hongcheng Sun,† Xiyu Zhang,† Lu Miao,‡ Linlu Zhao, Quan Luo, Jiayun Xu, and Junqiu Liu* State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China S Supporting Information *
ABSTRACT: Organic nanoparticle induced self-assembly of proteins with periodic nanostructures is a promising and burgeoning strategy to develop functional biomimetic nanomaterials. Cricoid proteins afford monodispersed and well-defined hollow centers, and can be used to multivalently interact with geometrically symmetric nanoparticles to form one-dimensional protein nanoarrays. Herein, we report that core-cross-linked micelles can direct cricoid stable protein one (SP1) to self-assembling nanowires through multiple electrostatic interactions. One micelle can act as an organic nanoparticle to interact with two central concaves of SP1 in an opposite orientation to form a sandwich structure, further controlling the assembly direction to supramolecular protein nanowires. The reported versatile supramolecular scaffolds can be optionally manipulated to develop multifunctional integrated or synergistic biomimetic nanomaterials. Artificial light-harvesting nanowires are further developed to mimic the energy transfer process of photosynthetic bacteria for their structural similarity, by means of labeling donor and acceptor chromophores to SP1 rings and spherical micelles, respectively. The absorbing energy can be transferred within the adjacent donors around the ring and shuttling the collected energy to the nearby acceptor chromophore. The artificial light-harvesting nanowires are designed by mimicking the structural characteristic of natural LH-2 complex, which are meaningful in exploring the photosynthesis process in vitro. KEYWORDS: self-assembly, protein nanowires, light-harvesting, core-cross-linked micelles, electrostatic interactions
P
limitation for the oriented assembly in a directed way to form anisotropic protein arrays.18,19 An alternative strategy is to accurately design the multivalent surface−surface interactions in manipulating self-assembling behavior of proteins, that is, fully considering the topological structure and surface characteristics of proteins. Stable protein one (SP1),20,21 a thermostable and electronegative protein, can bind together through hydrophobic interaction to create a double-layered homododecameric cricoid ring around a pseudo-6-fold axis (C6 symmetry) with an outer diameter of 11 nm, an inner diameter of 2.5 nm, and a height of about 4.5 nm (Scheme 1b). The surface potential analysis shows the acidic amino acids are mostly enriched in the top and the bottom surfaces of the cricoid structure, while alkaline amino acids are mainly focused on its inside and outside surfaces. Also, the symmetrical concave structure of the SP1 ring makes it possible that
roteins are the pivotal building blocks in nature for evolution of cellular machineries and molecular machines and for exploring the pathogenetic mechanisms of human disease.1−4 Despite their prevalence, little is known about the mechanisms that drive the formation and the assembly at the level of evolution in the cell.5 Scientists have inspired their great interests in disclosing the fundamental mechanism of protein self-assembly and further exploiting novel supramolecular architectures with hyperfine nanostructures by manipulating the protein−protein interactions accurately.6−10 Up to now, multifarious protein self-assemblies have been reported using different strategies, such as electrostatic interactions, host−guest interactions, metal-mediated interactions and so on.11−15 Due to their inherent charge distributions around the protein surface, electrostatic strategy is considered to be the most simple and effective approach to develop self-assembling protein nanostructures.16,17 Recently, binary protein complexes driven by multivalent electrostatic interactions have been successfully employed to develop multifunctional integrated or synergistic protein nanomaterials. However, these approaches have obvious © XXXX American Chemical Society
Received: August 20, 2015 Accepted: December 4, 2015
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Scheme 1. Structure-based design of protein nanowires. (a) The formation of core-crosslinked micelles (CCMs). (b) The surface topography of double-layered SP1 homododecameric cricoid structure. (c) Possible assembly model of one protein nanowire composed of eight SP1 rings (cyans) and seven CCMs (yellow)
cricoid structure of SP1 protein is similar to the bacterial lightharvesting (LH-2) complex.27 By labeling donor and acceptor chromophores to SP1 and spherical micelles, respectively, we further developed artificial light-harvesting system in vitro to mimic the energy transfer process in natural photosynthetic bacteria. The absorbing energy can be transferred within the adjacent donors around the ring and shuttle the collected energy to the nearby acceptor chromophores.
electropositive globular nanoparticles with diameter up to 2.5 nm may “sit” on the center of cricoid structure to further direct its “growth” to form one-dimensional protein nanoarrays.22 In our previous work, we have successfully demonstrated that an electropositively charged macromolecule, the fifth generation polyamidoamine dendrimer, is able to manipulate the selfassembly process of cricoid SP1 in a programmable manner to form nanowires.23 As predicted, one dendrimer can behave as an organic nanoparticle and can be stuck by two SP1 rings through surface−surface multiple electrostatic interactions along the C6 symmetry axis to form a sandwich structure, further controlling the assembly direction to supramolecular protein nanowires. Dendrimer, as a monodispersed macromolecule with well-defined architecture and molecular size, affords excellent nanoparticles characteristic and properties.24−26 However, it undergoes very complicated experimental procedures to produce a monodispersed dendrimer, and each intermediate product must be purified, which may greatly restrict the experimental practicability. We are curious about whether molecules with a comparable morphology to dendrimer, but possessing an easier preparation method and structural component can possibly control the self-assembly of proteins, which would greatly enrich the strategies in developing well-defined organic macromolecule−protein binary supramolecular hybrids. Surfactant micelles (Scheme 1a), affording a simple molecular structure and similar surface properties with dendrimers, may be the best and the simplest organic nanoparticle scaffolds. By taking advantage of their inherent structure, we employ electropositive surfactant micelles as organic nanoparticles, for the first time, to manipulate cricoid SP1 assembling to one-dimensional nanostructures. We anticipate that micelles could also afford nanoparticle properties as dendrimers, and proteins might be orderly arranged to nanowires with the protein−protein distance being restricted to several nanometers (Scheme 1c). The reported versatile supramolecular scaffolds can be optionally manipulated to develop multifunctional integrated or synergistic biomimetic nanomaterials. The dodecameric
RESULTS AND DISCUSSION The schematic illustration of surfactant micelles induced electrostatic self-assembly of cricoid protein into nanowires is shown in Scheme 1c. Cationic surfactant, (11acrylatylundecyl)triethylammonium bromide (see Supporting Information, Figure S1,S2), is able to self-assemble to micelles in aqueous solution when its concentration is higher than its critical micelle concentration, with hydrophilic quaternary ammonium groups dispersed around the surface.28 Because the acrylatyl groups are enriched in the core of micelles, the core-cross-linked micelles (CCMs) can be prepared by free radical polymerization in aqueous dispersion with the addition of an aqueous radical initiator, potassium persulfate (Scheme 1a). The 1H NMR spectrum of the CCMs showed that the acrylatyl peaks between 5.8 and 6.4 ppm nearly completely disappeared after reaction (see Supporting Information, Figure S3), indicating that the acrylatyl group was polymerized to a C−C single bond in the micelles. Dynamic light scatting (DLS) showed the average hydrodynamic diameter was nearly 10 nm, and it remained unchanged even under extremely low concentration (see Supporting Information, Figure S4). The CCMs afford considerable size and structural stability, which lays the foundation for the further development of well-defined protein−micelle binary hybrids to yield self-assembling protein nanoarrays through multivalent electrostatic interactions. The micelle-induced self-assembly process of SP1 was monitored by DLS and showed that the hydrodynamic diameter (Dh) of SP1/CCMs complexes increased to more than 120 nm when the CCMs content in SP1 aqueous solution B
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Figure 1. Morphologies of CCMs induced SP1 assemblies. (a) AFM image of the nanowires at the molar ratio of SP1/CCMs in 1:1. (b) Height profile along the black line in image a. (c) TEM image of SP1/CCMs nanowires. Inset is the model of the SP1/CCMs nanowire stacked with four SP1 rings and three CCMs. (d) TEM image of bundles of nanowires. The white arrows are directed to SP1 rings of SP1/ CCMs nanowires. Inset is the electrostatic interaction between quaternary amino groups (in green) of the CCMs and negative charges (Glu and Asp in red) around the outer surface of SP1 of another SP1/CCMs nanowire.
micelles could also control the self-assembling behavior of cricoid proteins to supramolecular nanowires through protein− micelle multivalent electrostatic interactions, which was exactly consistent with our previous expectation (Scheme 1c). We think that the proposed assembly strategy will provide suggestions in developing anisotropic protein nanomaterials and programmable protein delivery. We also found that protein nanowires could further stagger together along the horizontal or vertical directions to form bilayered or trilayered large-scale nanorods at high concentration (see Supporting Information, Figure S7). Bundles of nanowires through the alternative permutation were observed from the TEM image (Figure 1d) to further form staggered gear-like protein arrays (white arrow) because the exposed quaternary amino groups of CCMs could electrostatically interact with negative charges (Glu or Asp) around the SP1 outside surface of the adjacent nanowires (Figure 1d, inset).22,23 SP1 protein possesses a perfect stable cricoid-like structure, and it is similar to the natural LH-2 complex of photosynthetic bacteria. Octameric bacteriochlorophylls orderly arrange to a ring-like structure in the LH-2 complex with the distance of the nearest and the farthest bacteriochlorophylls to be 2.2 and 5.8 nm, respectively.27 We think we can mimic the energy transfer process of the natural LH-2 complex by site-directed design of chromophores to the SP1 ring. Computer simulation showed alanine residue (Ala 84) was exposed to the top and the bottom surfaces of SP1 ring with the nearest distance to be 3.1 nm and
was improved. Also, the effective length of the electrostatic interaction could be restricted by increasing the electrolyte strength. The surface charge of the SP1 and the micelles was shielded in high concentrated potassium chloride solution, which results in weak multivalent electrostatic interactions and the release of free proteins (see Supporting Information, Figure S5). Tapping-mode atomic force microscopy (AFM) and transmission electron microscope (TEM) are further employed to investigate the surface morphologies and the assembly behaviors. The AFM image indicated that the SP1/CCMs complexes possessed a generally linear structure with the length reaching up to 200 nm (Figure 1a,1b). The uniform height of the nanowires was 10.3 nm, quite consistent with the diameter (approximately 11 nm) of the SP1 crystal structure. The TEM image (Figure 1c) clearly showed SP1 rings stood face-to-face to form nanowires in SP1/CCMs hybrids, and the average ring−ring distance (5.3 nm) was larger than the crystal width (4.5 nm) of a single SP1 ring, suggesting that CCMs sandwiched SP1 rings along the C6 symmetry axis to form ordered one-dimensional protein nanowires. We further staked the model of SP1/CCMs nanoarrays composed of four repeating units for gaining greater structural observation (Figure 1c, inset). Different from the assembly morphologies, pure SP1 protein appeared as dimensional homogeneous dotlike nanostructures in the AFM image, and it afforded a monodispersed ring-like structure with the outer diameter about 11 nm in the TEM image (see Supporting Information, Figure S6). All these data supported our hypothesis that C
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Figure 2. Structure comparison of natural LH-2 complex (a) and chromophore modified SP1 (b).
Figure 3. (a) Donor (DPA) and acceptor (EY) chromophores used in this study. (b) Normalized absorption spectra (solid line) and fluorescence emission spectra (dash line) of DPA (yellow) and EY (rose).
energy transferred from DPA (Figure 4a). MALDI-TOF MS data showed the molecule weight of SP1-84Cys increased by the weight of the DPA after reaction (see Supporting Information, Figure S13), indicating that DPA was successfully labeled to the SP1 surface as put forth by our design. Most importantly, the chromophores labeled to the surface did not affect the self-aggregated behavior of protein, and DSP1 still retained its cricoid structure (see Supporting Information, Figure S14), which laid the foundation for further development of functional protein nanowires. The chromophores-labeled nanowires were constructed by ECCMs induced self-assembly of DSP1 using the same procedure as SP1/CCMs (Figure 4a). The TEM image showed that the introduction of chromophores to the building blocks did not affect the protein assembly morphologies (see Supporting Information, Figure S15). In other words, the supramolecular protein nanowires could effectively disperse donor and acceptor chromophores in close proximity to satisfy fluorescence resonance energy transfer.30−32 A significant decrease of fluorescence intensity of DSP1 could be observed after modifying two chromophores to the nanowires simultaneously (Figure 4b). Model analysis indicated a nanowire contained 225 donor chromophores per 100 nm in length, with an estimated spacing of 1.55 nm for DPA-to-DPA and 1.84 nm for DPA-to-EY. The gap between two nearest donor and acceptor chromophores can be drawn closer after micelle-
the farthest to be 6.1 nm. The Ala 84 site was mutated to a reactive cysteine residue (Cys 84) for chromophore attachment (Figure 2). After expression and purification of the mutant protein (SP1-84Cys) in Escherichia coli, the desired protein was confirmed by SDS-PAGE analysis, CD spectra, and matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis (see Supporting Information, Figures S8−S10). The thiol-reactive chromophore, 9-[4-(bromomethyl)phenyl]-10-(4-methylphenyl)anthracene (DPA-Br) is selected for this study as donor chromophore (see Supporting Information, Figure S11, S12). Eosin Y disodium salt (EY) is also employed as acceptor chromophore due to the overlap of its absorption with DPA emission.29 As shown in Figure 3, DPA emits a fluorescence band between 400 and 500 nm with an excitation light at 375 nm. In addition, EY presents an emission band centered at 550 nm with an excitation wavelength at 490 nm. Appreciable spectrum overlap of DPA absorption and emission allows the transfer of fluorescence resonance energy among adjacent donors, also the overlap between DPA emission and EY absorption benefit the energy transfer from donor-to-acceptor. On one hand, DPA-modified SP1 (DSP1) was successfully prepared by a thiol−halogen click-like reaction of SP1-84Cys and DPA-Br employing a strong base. On the other hand, EY was labeled to the surface of CCMs (ECCMs) by electrostatic interaction to receive the D
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Figure 4. Design of light-harvesting nanowires. (a) Assembly model of DSP1/ECCMs nanowire with DSP1 (cyans) and ECCMs (yellow). Donor, DPA (purple), is designed on the top and bottom surfaces of SP1. Acceptor, EY (rose), is modified on the surface of CCMs by electrostatic interactions. (b) Fluorescence spectra of DSP1/CCMs and DSP1/ECCMs upon excitation at 375 nm. (c) For systems with large numbers of donors, energy can be transferred to acceptor chromophores via direct donor-to-energy transfer (Path 1) or multistep donor-todonor transfers (Path 2).
to ECCMs was increased, indicating DPA could not transfer its absorbed energy to ECCMs (see Supporting Information, Figure S16b). All these data implied that the absorbing energy can be successfully transferred in the artificial light-harvesting nanowires. To evaluate the light-harvesting capability of the DSP1/ ECCMs complexes, it is necessary to evaluate the overall energy transfer efficiency (E).34,35 For a single-donor/single-acceptor system, E is easily calculated from the simplified Förster theory:
induced protein self-assembly. We anticipate that the absorbing energy can be transferred within the adjacent donors around the ring and shuttle the collected energy to the nearby acceptor chromophore (Figure 4c). The artificial light-harvesting nanowires were designed by mimicking the structural characteristic of the natural LH-2 complex, which is meaningful in exploring the photosynthesis process in vitro. With an improvement of the EY content in DSP1/ECCMs complexes, the emission intensity at 549 nm was enhanced and that at 435 nm was gradually attenuated when excited at 375 nm. The attenuation of DSP1 emission was due to the reason that the absorbing energy was transferred from DSP1 to ECCMs (Figure 5a). For comparison, EY was added to pure DSP1 solution to investigate the energy transfer process. The emission intensity at 549 nm was increased but it remained unchanged at 435 nm because the absorbing energy of DSP1 could not be transferred to EY (see Supporting Information, Figure S16a). These results showed that ECCMs could accept energy transferred from DSP1 after the assembly of proteins and micelles. The ratio of emission intensity at 549 and 435 nm (I549/I435) was employed to evaluate the light harvesting ability.33 It was found that the ratio value of I549/I435 was significantly magnified when the acceptor content was improved (Figure 5b). On the contrary, the improvement of DSP1 content afforded both enhancement of emission intensity for the donor at 435 nm and acceptor at 549 nm. Although the content of ECCMs was not increased, the ratio values of I549/ I435 were almost unchanged (Figure 5c,d). However, the emission of acceptor was not enhanced when the DPA content
E=
3 2 6 k R0 2 3 2 6 k R0 + 2
r6
(1)
In the above equation, R0 is the dynamically averaged Förster radius at which the energy transfer from donor to acceptor is at 50% efficiency; r and k are the actual donor−acceptor distance and the orientation factor, and k2 is equal to 2/3 for the randomly distributed chromophores. The simplified equation shows that the transfer efficiency is only dependent on the ratio of r and R0. r can be measured from the ideal assembly model. R0 can also be calculated from the following equation: R 0 = 0.211(k 2η−4 Q DJ(λ))1/6 (in Å)
(2)
where η, QD, and J(λ) are the aqueous refractive index (η = 1.33), the donor quantum yield, and the integrated spectral overlap of normalized donor fluorescence and acceptor absorption coefficient, respectively. QD is estimated by E
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Figure 5. Fluorescence properties of electrostatically induced DSP1 assemblies with ECCMs. (a) FRET spectra of DSP1/ECCMs with various amounts of EY upon excitation at 375 nm ([DPA] = 10 μM). (b) The fluorescence intensities of DSP1/ECCMs at 549 and 435 nm versus EY concentrations. (c) FRET spectra of DSP1/ECCMs with various amount of DPA upon excitation at 375 nm ([EY] = 2.0 μM). (d) The fluorescence intensities of DSP1/ECCMs at 549 and 435 nm versus DPA concentrations. Each experiment was repeated for three times.
Table 1. Energy Transfer Parameters of Donor−donor and Donor−acceptor Pairs of DSP1/ECCMs Nanowires. Each Experiment Was Repeated Three Times chromophores
QD (×10−2)
J(λ) (×1013·M−1·cm−1·nm4)
R0 (nm)
r (nm)
E (%)
DPA−DPA DPA−EY
5.2 ± 0.2 5.2 ± 0.2
2.1 ± 0.1 4.4 ± 0.3
1.65 ± 0.03 1.87 ± 0.03
1.55 1.84
59 ± 3 52 ± 2
QD = Q s ×
ID η2 × 2 Is ηs
transfer efficiency of donor-to-donor and donor-to-acceptor at the same time. The overlap integral J(λ) of DPA−DPA and DPA−EY was calculated to be (2.1 ± 0.1) × 1013 and (4.4 ± 0.3) × 1013 M−1·cm−1·nm4, and the Förster radius R0 was thus estimated to be 1.65 ± 0.03 and 1.87 ± 0.03 nm, respectively. The actual r values were measured to be 1.55 and 1.84 nm by the staking model for adjacent donor−donor and donor− acceptor pairs. Therefore, the value of E was calculated by eq 1 to be 52% for the energy transfer from DPA to EY (Path 1, Figure 4c) and to be 59% for the adjacent donors (Path 2, Figure 4c). We further investigated the lifetime of the donor and showed significant change in the absence and in the presence of the acceptor (see Supporting Information, Figure S17). The average lifetime (⟨τ⟩) of DSP1 was 3.06 ns, but it was modified to 1.60 ns in the presence of ECCMs, which also indicated the possibility of energy transfer from DPA to EY in the DSP1/ECCMs solution. All these data showed that our developed artificial light-harvesting antenna could transfer absorbing energy around the SP1 ring and then transport to the nearby EY with high energy transfer efficiency. The artificial light-harvesting nanowires were designed by mimicking the
(3)
where Qs, Is, and ηs are the quantum yield, intergrated intersity, and refractive index of the standard sample. ID and ηD are the intergrated intensity and refractive index of donor. QD was tested to be 0.052. J(λ) was further calculated using the following equation: J (λ ) =
∫0
∞
fD (λ) εA (λ) λ 4 dλ
(4)
where λ, εA(λ) and f D(λ) are the wavelength of light (nm), the molar absorptivity of the acceptor and the normalized donor fluorescence spectrum. εA(λ) was calculated by the absorption spectrum of acceptor under certain concentration. f D(λ) was normalized on the wavelength scale according to 1=
∫0
∞
fD (λ) dλ
(5)
The absorbing energy by DSP1 could not only be transferred to the nearby ECCMs, but also transferred within the adjacent donors (Table 1). It is meaningful to measure the energy F
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collect the water solution. The mixture was purified by Sephadex G25 and freeze-dried to get solid powder. Self-Assembly of SP1/CCMs Nanowires. The self-assembly process of SP1/CCMs nanowires was as follows: SP1 was dissolved in Milli-Q with the final concentration reaching to 25 mg/L. A 500 μL sample of SP1 solution was mixed with 500 μL of fresh CCMs (2.0 mg/L) at 4 °C and underwent ultrasound for 10 min. The mixture was left to stand for 30 min before use. Dynamic Light Scatting. Dynamic light scattering (DLS) experiments were carried out with Malvern Instrument Zetasizer Nano ZS. An 800 μL sample of SP1/CCMs complexes with the final SP1 concentration of 12.5 mg/L was added to the sample cube to monitor the hydrodynamic diameter. The final concentration of CCMs was 0 mg/L, 0.10 mg/L, 0.25 mg/L, 0.50 mg/L, and 1.0 mg/L for every complex. At least five measurements were tried to reach an average value. Atomic Force Microscopy. Typical tapping-mode atomic force microscopy (AFM) measurements were performed using NanoScope Multimode AFM (Veeco, USA). SP1/CCMs complexes for AFM images were prepared by dispersing the sample onto a freshly hydrogen-implanted (111) silicon wafer and allowing it to dry in air. Transmission Electron Microscopy. Transmission electron microscopy (TEM) images of SP1/CCMs were recorded at an acceleration voltage of 200 kV on a JEM-2100F microscope (JEOL Ltd.). TEM samples were prepared by spreading a droplet of diluted SP1/CCMs solution on standard carbon-coated Formvar films on copper grids for 10 min. The excess solution was blotted away with filter paper. The samples were further negatively stained with 4% sodium phosphotungstate aqueous solution for 40 s. The samples were dried in air overnight before use.
structural characteristic of the natural LH-2 complex, which was meaningful in exploring the photosynthesis process in vitro.
CONCLUSION In summary, micelle-induced protein nanowires were developed, for the first time, through electrostatic self-assembly of electronegative cricoid SP1 proteins and positively charged core-cross-linked micelles (CCMs). Micelles could also behave as organic nanoparticles in controlling the protein self-assembly process. Atomic force microscopy images and transmission electron microscopy images showed that CCM can be stuck by two cricoid SP1 rings through surface multiple electrostatic interactions at the central concaves to form sandwich structures, further controlling the assembly direction to supramolecular protein nanowires. Furthermore, at higher concentration, the protein nanowires were able to stagger together by the side of the electrostatic interactions to form large-scale nanorods. We also designed artificial light-harvesting nanowires to mimic the energy transfer process of photosynthetic bacteria by means of labeling donor (DPA) and acceptor (EY) chromophores to SP1 and spherical micelles, respectively. The fluorescence spectra and lifetime measurement showed that the absorbing energy could be transferred within the adjacent donors around the ring and the collected energy could be shuttled to the nearby acceptor chromophore for the DSP1/ECCMs nanowires. Therefore, the reported versatile supramolecular scaffolds can be optionally manipulated to develop different multifunctional integrated or synergistic biomimetic nanomaterials by the introduction of functional segments to proteins and organic molecules.
ASSOCIATED CONTENT S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b05213. Detailed synthesis procedures and characterization (1H NMR) of CCMs and DPA-Br; expression and characterization (SDS-PAGE, CD spectra, and MALDI-TOF analyses) of mutant SP1−84Cys; MALDI-TOF mass spectrometry of DSP1; dynamic light scattering (DLS) and atomic force microscopy (AFM) of the SP1/CCMs complexes; TEM images of SP1 and chromophoreslabeled DSP1/ECCMs; fluorescence resonance energy transfer process, and the lifetime of the DPA (PDF)
EXPERIMENTAL SECTION Preparation of CCMs. (11-Acrylatylundecyl)triethylammonium bromide (170 mg, 0.42 mmol) was dissolved in 10 mL of water and degassed with nitrogen for 30 min. Then radical initiator of free radical polymerization, potassium persulfate (5 mg), was added to the mixture and incubated at 60 °C for 72 h. After the reaction, the mixture was injected into a dialysis tube (Spectra/Pro Membrane, MWCO = 3500) for 3 days to remove the oligomers and radical initiator. Construction and Expression of SP1-84Cys Plasmids. The SP1-84Cys gene was achieved by site-directed mutation using a sense primer with the sequence 5′-CTCGATTCTGCTTGTCTTGCTGCATTT-3′ and an antisense primer with the sequence 5′GCAAATGCAGCAAGACAAGCAGAATC-3′. An expression plasmid of pet22b-SP1−84Cys was extracted from bacterial strain DH5α. The construction of SP1-84Cys mutant was made using standard recombinant techniques. The plasmid with a SP1-84Cys domain was transformed into an Escherichia coli strain BL21. The LB culture was shaken at 37 °C until the OD600 value reached 0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 1.0 mM to induce expression. Cultures were grown 5 h at 28 °C, harvested by centrifugation, and stored at −20 °C. Induced cells were thawed and resuspended in Tris-HCl buffer (50 mM, pH = 6.3) containing 1.0 mM PMSF and 50 mM NaCl. Cells were ultrasonicated for 10 min and heated and centrifuged to remove the precipitations. The crude protein solution was further purified by DEAE ion-exchange column and Sephadex G75 to remove the impurity proteins and nucleic acids. Design of DPA-Labeled SP1 (DSP1). DPA-labeled SP1 (DSP1) was successfully prepared by the thiol−halogen click-like reaction of SP1-84Cys and DPA-Br. SP1-84Cys (10 mg) and DPA-Br (1 mg) were dissolved in 2 mL of DMSO. Then triethylamine (20 μL) was added to the mixture under uniform stirring to support the alkaline environment. After reaction for 24 h at room temperature, 10 mL of Milli-Q was added to the reaction, and the mixture was filtrated to
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Present Address ‡
L.M.: Key Laboratory of Separation Science and Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China Author Contributions †
H.S. and X.Z. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS We thank Prof. Oded Shoseyov from the Hebrew University of Jerusalem for his kind supply of the SP1 gene. This work was also supported by the National Natural Science Foundation of China (No: 21234004, 21420102007, 21221063, 21574056), G
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ACS Nano
(21) Medalsy, I.; Dgany, O.; Sowwan, M.; Cohen, H.; Yukashevska, A.; Wolf, S. G.; Wolf, A.; Koster, A.; Almog, O.; Marton, I.; et al. SP1 Protein-Based Nanostructures and Arrays. Nano Lett. 2008, 8, 473− 477. (22) Miao, L.; Han, J. S.; Zhang, H.; Zhao, L. L.; Si, C. Y.; Zhang, X. Y.; Hou, C. X.; Luo, Q.; Xu, J. Y.; Liu, J. Q. Quantum-Dot-Induced Self-Assembly of Cricoid Protein for Light Harvesting. ACS Nano 2014, 8, 3743−3751. (23) Sun, H. C.; Miao, L.; Li, J. X.; Fu, S.; An, G.; Si, C. Y.; Dong, Z. Y.; Luo, Q.; Yu, S. J.; Xu, J. Y.; et al. Self-Assembly of Cricoid Proteins Induced by “Soft Nanoparticles”: An Approach to Design Multienzyme-Cooperative Antioxidative Systems. ACS Nano 2015, 9, 5461−5469. (24) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. A New Class of Polymers: Starburst-Dendritic Macromolecules. Polym. J. 1985, 17, 117−132. (25) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A., III Starburst Dendrimers: Molecular-Level Control of Size, Shape, Surface Chemistry, Topology, and Flexibility from Atoms to Macroscopic Matter. Angew. Chem., Int. Ed. Engl. 1990, 29, 138−175. (26) Esfand, R.; Tomalia, D. A. Poly (amidoamine) (PAMAM) Dendrimers: From Biomimicry to Drug Delivery and Biomedical Applications. Drug Discovery Today 2001, 6, 427−436. (27) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Crystal Structure of an Integral Membrane Light-Harvesting Complex from Photosynthetic Bacteria. Nature 1995, 374, 517−521. (28) Cui, X. H.; Mao, S. Z.; Liu, M. L.; Yuan, H. Z.; Du, Y. R. Mechanism of Surfactant Micelle Formation. Langmuir 2008, 24, 10771−10775. (29) Peng, H. Q.; Chen, Y. Z.; Zhao, Y.; Yang, Q. Z.; Wu, L. Z.; Tung, C. H.; Zhang, L. P.; Tong, Q. X. Artificial Light-Harvesting System Based on Multifunctional Surface-Cross-Linked Micelles. Angew. Chem., Int. Ed. 2012, 51, 2088−2092. (30) Miller, R. A.; Presley, A. D.; Francis, M. B. Self-Assembling Light-Harvesting Systems from Synthetically Modified Tobacco Mosaic Virus Coat Proteins. J. Am. Chem. Soc. 2007, 129, 3104−3109. (31) Miller, R. A.; Stephanopoulos, N.; McFarland, J. M.; Rosko, A. S.; Geissler, P. L.; Francis, M. B. Impact of Assembly State on the Defect Tolerance of TMV-Based Light Harvesting Arrays. J. Am. Chem. Soc. 2010, 132, 6069−6074. (32) Dedeo, M. T.; Duderstadt, K. E.; Berger, J. M.; Francis, M. B. Nanoscale Protein Assemblies from a Circular Permutant of the Tobacco Mosaic Virus. Nano Lett. 2010, 10, 181−186. (33) Pu, F.; Wu, L.; Ju, E. G.; Ran, X.; Ren, J. S.; Qu, X. G. Artificial Light-Harvesting Material Based on Self-Assembly of Coordination Polymer Nanoparticles. Adv. Funct. Mater. 2014, 24, 4549−4555. (34) Montali, A.; Harms, G. S.; Renn, A.; Weder, C.; Smith, P.; Wild, U. P. Time-Resolved Fluorescence Study on the Mechanism of Polarizing Energy Transfer in Uniaxially Oriented Polymer Blends. Phys. Chem. Chem. Phys. 1999, 1, 5697−5702. (35) Lunz, M.; Bradley, A. L.; Gerard, V. A.; Byrne, S. J.; Gun’ko, Y. K.; Lesnyak, V.; Gaponik, N. Concentration Dependence of Förster Resonant Energy Transfer Between Donor and Acceptor Nanocrystal Quantum Dot Layers: Effect of Donor-Donor Interactions. Phys. Rev. B: Condens. Matter Mater. Phys. 2011, 83, 115423−115423.
111 Project (B06009) and the Chang Jiang Scholars Program of China.
REFERENCES (1) Ferrer, M.; Golyshina, O. V.; Beloqui, A.; Golyshin, P. N.; Timmis, K. N. The Cellular Machinery of Ferroplasma Acidiphilum is Iron-Protein-Dominated. Nature 2007, 445, 91−94. (2) Bale, S. S.; Kwon, S. J.; Shah, D. A.; Banerjee, A.; Dordick, J. S.; Kane, R. S. Nanoparticle-Mediated Cytoplasmic Delivery of Proteins To Target Cellular Machinery. ACS Nano 2010, 4, 1493−1500. (3) Roychaudhuri, R.; Yang, M. F.; Hoshi, M. M.; Teplow, D. B. Amyloid beta-Protein Assembly and Alzheimer Disease. J. Biol. Chem. 2009, 284, 4749−4753. (4) Li, H. B.; Cao, Y. Protein Mechanics: From Single Molecules to Functional Biomaterials. Acc. Chem. Res. 2010, 43, 1331−1341. (5) Levy, E. D.; Erba, E. B.; Robinson, C. V.; Teichmann, S. A. Assembly Reflects Evolution of Protein Complexes. Nature 2008, 453, 1262−1265. (6) Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H. DNA-Templated Self-Assembly of Protein Arrays and Highly Conductive Nanowires. Science 2003, 301, 1882−1884. (7) Rammensee, S.; Slotta, U.; Scheibel, T.; Bausch, A. R. Assembly Mechanism of Recombinant Spider Silk Proteins. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 6590−6595. (8) Levy, Y.; Onuchic, J. N. Mechanisms of Protein Assembly: Lessons from Minimalist Models. Acc. Chem. Res. 2006, 39, 135−142. (9) Fegan, A.; White, B.; Carlson, J. C. T.; Wagner, C. R. Chemically Controlled Protein Assembly: Techniques and Applications. Chem. Rev. 2010, 110, 3315−3336. (10) Carlson, J. C. T.; Jena, S. S.; Flenniken, M.; Chou, T. F.; Siegel, R. A.; Wagner, C. R. Chemically Controlled Self-Assembly of Protein Nanorings. J. Am. Chem. Soc. 2006, 128, 7630−7638. (11) Kostiainen, M. A.; Hiekkataipale, P.; Laiho, A.; Lemieux, V.; Seitsonen, J.; Ruokolainen, J.; Ceci, P. Electrostatic Assembly of Binary Nanoparticle Superlattices Using Protein Cages. Nat. Nanotechnol. 2013, 8, 52−56. (12) Hou, C. X.; Li, J. X.; Zhao, L. L.; Zhang, W.; Luo, Q.; Dong, Z. Y.; Xu, J. Y.; Liu, J. Q. Construction of Protein Nanowires through Cucurbit[8]uril-based Highly Specific Host−Guest Interactions: An Approach to the Assembly of Functional Proteins. Angew. Chem., Int. Ed. 2013, 52, 5590−5593. (13) Bai, Y. S.; Luo, Q.; Zhang, W.; Miao, L.; Xu, J. Y.; Li, H. B.; Liu, J. Q. Highly Ordered Protein Nanorings Designed by Accurate Control of Glutathione S-Transferase Self-Assembly. J. Am. Chem. Soc. 2013, 135, 10966−10969. (14) Pippig, D. A.; Baumann, F.; Strackharn, M.; Aschenbrenner, D.; Gaub, H. E. Protein-DNA Chimeras for Nano Assembly. ACS Nano 2014, 8, 6551−6555. (15) Niu, Z. W.; Liu, J.; Lee, L. A.; Bruckman, M. A.; Zhao, D. G.; Koley, G.; Wang, Q. Biological Templated Synthesis of Water-Soluble Conductive Polymeric Nanowires. Nano Lett. 2007, 7, 3729−3733. (16) Perutz, M. F. Electrostatic Effects in Proteins. Science 1978, 201, 1187−1191. (17) Beck, T.; Tetter, S.; Kuenzle, M.; Hilvert, D. Construction of Matryoshka-Type Structures from Supercharged Protein Nanocages. Angew. Chem., Int. Ed. 2015, 54, 937−940. (18) Moerz, S. T.; Kraegeloh, A.; Chanana, M.; Kraus, T. Formation Mechanism for Stable Hybrid Clusters of Proteins and Nanoparticles. ACS Nano 2015, 9, 6696−6705. (19) Kostiainen, M. A.; Kasyutich, O.; Cornelissen, J. J. L. M.; Nolte, R. J. M. Self-Assembly and Optically Triggered Disassembly of Hierarchical Dendron−Virus Complexes. Nat. Chem. 2010, 2, 394− 399. (20) Dgany, O.; Gonzalez, A.; Sofer, O.; Wang, W. X.; Zolotnitsky, G.; Wolf, A.; Shoham, Y.; Altman, A.; Wolf, S. G.; Shoseyov, O.; Almog, O. The Structural Basis of the Thermostability of SP1, a Novel Plant (Populus Tremula) Boiling Stable Protein. J. Biol. Chem. 2004, 279, 51516−51523. H
DOI: 10.1021/acsnano.5b05213 ACS Nano XXXX, XXX, XXX−XXX
