Template-Free Construction of Highly Ordered Monolayered

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Template-Free Construction of Highly Ordered Monolayered Fluorescent Protein Nanosheets: A Bio-Inspired Artificial Light-Harvesting System Xiumei Li, Shanpeng Qiao, Linlu Zhao, Shengda Liu, Fei Li, Feihu Yang, Quan Luo, Chunxi Hou, Jiayun Xu, and Junqiu Liu ACS Nano, Just Accepted Manuscript • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019

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Template-Free Construction of Highly Ordered Monolayered Fluorescent Protein Nanosheets: A Bio-Inspired Artificial Light-Harvesting System Xiumei Li, Shanpeng Qiao, Linlu Zhao, Shengda Liu, Fei Li, Feihu Yang, Quan Luo, Chunxi Hou, Jiayun Xu, Junqiu Liu* State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China *Address correspondence to: [email protected]

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ABSTRACT

Using biological materials for light-harvesting applications has attracted considerable attention in recent years. Such materials provide excellent environmental compatibility and often exhibit superior properties over synthetic materials. Herein, inspired by the outstanding energy transfer performance in coelenterates, we constructed a template-free, highly ordered 2D light-harvesting system by covalent-induced co-assembly of EBFP2 (donor) and EGFP (acceptor), in which the fluorescent chromophores were well distributed and adopted a fixed orientation. By introducing approximate square planar binding sites on the side surface of protein, assembly pattern was pin down and self-assembly extended in orthogonal directions to achieve monolayered and tessellated protein nanoarrays. The excellent anti-self-quenching property of fluorescent proteins endowed the co-assembled system with attractive light-harvesting capability. Even at high local concentrations, a low resonance energy transfer self-quenching was observed, therefore, energy can be efficiently transferred. More importantly, the distance between adjacent chromophores is continuous adjustable. By making minor changes to the length of the inducing linker, we have achieved significant control over the size of the assembly. A micron-sized light-harvesting system with satisfactory energy transfer efficiency was finally obtained. This work developed a template-free light-harvesting system completely based on fluorescent proteins (FPs), which overcame the restriction of using templates. Not limited to this work, the special core-shell structure of FPs may be expected to direct the optimization of fluorescent dyes by cladding.

KEYWORDS: protein assembly, protein nanosheets, EBFP2, EGFP, light-harvesting, covalent interaction


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The natural photosynthesis system is considered to be one of the most elaborate nanobiological machine. It is a huge material and energy conversion station, which provides the material and energy foundation for all living organisms on the earth. Marveling at the precision and the efficiency of the natural photosynthetic system, a variety of natural light-harvesting mimics were built in recent years.1,2 However, most of the fluorescent molecules are limited in the application of light-harvesting systems due to the “aggregation-caused quenching” (ACQ). To avoid the undesired self-quenching, chromophores need to be separated enough to minimize self-quenching while ensuring the efficient energy transfer from donor to acceptor. Therefore, most of the fluorescent molecules in artificial light-harvesting systems need to be arranged with the help of templates such as organic-inorganic hybrid materials,3,4 dendrimers,5,6 nanomicells,7,8 organogels9,10 and biomaterials.11-13 Although the using of templates can provide a strategy to eliminate self-quenching, it remains difficult to achieve high local densities of chromophores without undesired self-quenching and the sparse chromophore binding sites do limit the improvement of energy transfer efficiency. During the long-term evolutionary process, biologically produced materials are often endowed with superior properties over synthetic materials. Using these bio-optimized materials can often easily overcome the shortcomings of synthetic materials and receive unexpected results. In the deep ocean, green light emission is observed in many coelenterates as a result of a non-radiative energy transfer from luciferase to the green fluorescent protein. There is evidence that to have such an efficient energy migration in bioluminescent coelenterates, luciferase and green fluorescent protein (GFP) must have a sufficiently high concentration and may need in to be orderly and tightly arranged.14 This reminds us that the bio-optimized fluorescent protein may


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have an excellent anti-self-quenching property and hold great promise for light-harvesting applications. GFP was evolved to be the energy acceptor in several bioluminescent coelenterates. Ever since its discovery, GFP and its derivatives have been widely used as a powerful tool for exploring biological structure and function. Förster resonance energy transfer (FRET) technique based on fluorescent proteins FRET pairs is widely used for designing fluorescent biosensors to probe the cellular environment and to visualize protein-protein interactions in situ.15-18 GFP has a nearly perfect beta-barrel structure with the chromophore located in the center.19,20 The tightly packed βstrands isolate the chromophores from the external environment and prevent the chromophore fluorescence from quenching by water. What is more, the tightly packed protective shells prevent the close interaction of adjacent fluorophores effectively and minimize self-quenching even in dense aggregation, which shows a great advantage over the organic dyes whose dense aggregation would result in excessive quenching.21,22 Thus, a template-free artificial lightharvesting system is possible to be constructed completely based on the FPs. The rapid development of protein self-assembly science provides the idea for constructing such a template-free bio-inspired light-harvesting system. Over the past few decades, great efforts have been devoted to the design and construction of highly ordered protein assemblies. A variety of regularly organized protein nanostructures including fibers, helices, tubes, rings and cages are constructed successfully via several strategies.23,24 Various driving forces such as covalent interaction25-27 and non-covalent interactions including metal coordination,28-30 host-guest interaction,31-33 electrostatic interaction,34-36 receptor-ligand interaction37,38 are employed to manipulate protein to self-assemble into highly ordered nanostructures. The rich protein selfassembly strategies provide a facile means for organizing a large number of FPs into dense and


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highly ordered supramolecular nanostructures. Not limited to proteins with special properties, covalent interaction provides a simple and universal approach to organize proteins in an orderly manner. Given the fact that cysteine is a rare amino acid, covalent cross-linking rely on cysteine residue can occur at any desired location on the protein surface. More importantly, the distance between monomers can be continuously adjusted by changing the length of linker, which is very meaningful for the construction and research of light-harvesting system. Therefore, covalent interaction can be an ideal driving force for constructing such a stable biological light-harvesting system. Herein, we reported a 2D FPs-based light-harvesting system with the help of covalent-induced protein self-assembly rather than templates. EBFP2 and EGFP were selected as the energy donor and acceptor respectively. As an improved version, EBFP2 was reported to be 4-fold brighter and 550-fold more photostable than EBFP, which makes EBFP2 more suitable as the donor of EGFP than its predecessor.39 The adjacent monomers were directly covalently cross-linked by bifunctional linkers to form highly ordered and monolayered protein nanosheets, in which, energy transferred efficiently from donor to acceptor with almost no self-quenching. The covalent cross-linking between adjacent monomers greatly improved the stability of the protein assemblies and overcame the restriction of using templates. Furthermore, in order to obtain large-scaled assemblies, we synthesized the cross-linkers with different end-to-end distances. A minor change in the length of the inducing linker exhibited a significant effect on the size of the assemblies. By using EG3DM as linker, a micron-sized light-harvesting system was finally obtained. RESULTS AND DISCUSSION


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Two-dimensional (2D) nanosheet can be an ideal model for light harvesting as it can densely arrange chromophores in a fixed orientation and distance and provide multiple energy transfer paths. From a retrosynthetic perspective, a highly ordered 2D sheet can be constructed by selfassembling C4 symmetric building blocks in orthogonal directions. Therefore, as a start point of constructing a 2D light-harvesting system, we first constructed EBFP2 and EGFP variants by introducing four cysteine lying near the C4 rotation axes at the lateral surface of FP, which were hereafter referred to as EBFP2-4C and EGFP-4C, respectively. To ensure site-specific crosslinking, we first removed the native cysteine (C48) in the surface of EBFP2 and EGFP. After carefully investigating the protein surface, four approximate square planar binding sites (E32-E124-K166-Q204) on the side of the beta-barrel were screened and then mutated into cysteine (Figure 1a, Figure S4), thus, the assembly pattern was pin down and the self-assembly is able to proceed along orthogonal directions. Meanwhile, the design of cross-linker is also crucial: short linker is in favor of energy transferring but is not conducive to achieve large-scaled assemblies due to the steric hindrance and the electrostatic repulsion between adjacent monomers. In order to obtain large-scaled protein assemblies with satisfactory energy transfer efficiency, a series of thiol-reactive linkers with different end-to-end distances were designed and synthesized (Figure 1b). The PEG chain between the two maleimides “heads” prevented the steric clashes and ensured the water solubility of the molecules. As depicted in Figure 1c, the self-assembly behavior was triggered upon addition of cross-linker. EBFP2-4C and EGFP-4C were packed together shoulder to shoulder and grew horizontally to form long-range order 2D monolayered protein nanoarrays ultimately.


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Figure 1. (a) Cartoon representation of EGFP-4C from the side view and the top view. The side chains of the four cysteine on the side of the beta-barrel were labeled with white spheres. (b) Thiol-reactive linkers with different end-to-end distances. Maleimide "head" and intermediate PEG chains were colored with yellow and red respectively. (c) Schematic representation of the formation of tessellated protein nanosheets via covalentinduced self-assembly. Negatively charged aspartic acid and glutamic acid at the interface were represented by white.

The EBFP2-4C and EGFP-4C variants were expressed in Escherichia coli strain and subsequently purified by Ni-NTA system. The purity of the proteins was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight was determined by MALDI-TOF mass spectrometry (Figure S5). The verified building blocks were then used for the construction of light-harvesting system. Considering the high reactivity between thiol and maleimide, the co-assembly was carried out at 4 ºC to slow down the assembly


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dynamics and ensure the long-range order of assemblies. After testing various assembly ratios (cross-linker:protein), the 3:1 ratio was determined to be the optimum proportion for assembly. The resulting building blocks EBFP2-4C and EGFP-4C were mixed at a molar ratio of 3:1 to a final concentration of 12 μM and then allowed to react with three different-length linkers in TrisHCl buffer (10 mM Tris-HCl, 20 mM NaCl, pH = 7.4) overnight. The formation of protein assemblies was initially verified by Dynamic light scattering (DLS) (Figure S8). Tapping-mode atomic force microscopy (AFM) and transmission electron microscope (TEM) were subsequently employed to investigate the morphology of assemblies. As shown in Figure 2, 2D monolayered nanosheets were successfully produced. The uniform height of the nanosheets was 4.5±0.2 nm, which was consistent with the height of the side surface of fluorescent protein (approximately 4.2 nm) but significantly higher than the diameter of the fluorescent protein (2.4 nm), whence we inferred that the FPs in the nanosheets were arranged shoulder to shoulder as intended. TEM data (Figure 2g) have also well supported this inference. The high magnification TEM image clearly showed that proteins were closely organized and extend in two directions to form tessellated 2D nanosheets, which is consistent with our design. Notably, the size of the assemblies induced by EG2DM and EG3DM exhibited a distinct difference: the EG3DM-induced assemblies (Figure 2c, 2f) showed a significantly larger size compared with those induced by EG2DM (Figure 2a). A proposed explanation for the difference in size is that long linker is advantageous for the formation of large-scaled assemblies by reducing the steric hindrance and the electrostatic repulsion between adjacent monomers. Therefore, only a few hundred nanometers of 2D layered nanostructures were observed when induced by a shorter linker (EG2DM), whereas a slight increase in the length of linker allowed the formation of micron-sized 2D nanosheets. However, the longer and more flexible EG4DM may be disadvantageous for organizing proteins to grow only along the horizontal, thus, limited the formation of larger assemblies. When assembling with


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EG4DM, FPs were prone to assembling in a random manner to form small aggregates (Figure S10). These results demonstrated that the size of assemblies was controllable via a minor change in the length of the inducing linker. A size controllable protein self-assembly system was constructed by changing the length of linker.


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Figure 2. Morphology characterization of the self-assembly protein nanosheets with different linkers (EG2DM and EG3DM). (a) AFM image of the 2D layered protein architectures assembled by FPs with 3 equiv of EG2DM. (b) Height profile along the black line in (a). (c) AFM image of the 2D layered protein architectures assembled by FPs with 3 equiv of EG3DM after purified by HPLC. (d) 3D image of (c). (e) Height profile along the black line in (c). (f) TEM image of FPs/EG3DM nanosheet. (g) The enlarged TEM image of FPs/EG3DM nanosheet. Inset: Schematic presentation of the packing mode of proteins.

To probe whether the observed planar sheets were properly co-assembled from EBFP2-4C and EGFP-4C as designed, fluorescence emission spectroscopy was employed to elucidate the coassembly mode (Figure 3a). As shown in Figure 3a, the addition of linkers resulted in a pronounced energy transfer from the donor EBFP2-4C to the acceptor EGFP-4C. There was an obvious decrease in the emission intensity of the donor and a clear increase in the emission intensity of the acceptor, which is a strong evidence of the co-assembly of EBFP2-4C and EGFP4C. In addition, when reacted with EG4DM, EG3DM and EG2DM, respectively, systems showed obvious differences in energy transmission. FPs/EG2DM and FPs/EG3DM shared a similar fluorescence spectra, and both exhibited a stronger energy transfer capacity than FPs/EG4DM. A visible color change from blue (before assembly) to green (after assembly) was also observed directly under UV light, which also strongly proved the co-assembly behavior. Furthermore, to confirm the assembly process was driven by covalent interaction, the assembly samples were directly analyzed by 12% SDS-PAGE. As shown in Figure 3b, compared with control, a new band with higher molecular weight was observed at the junction of stacking gel and separation gel, which indicated the formation of large protein complex and validated the covalent crosslinking between proteins. In addition, the dim tracks in stacking gel indicated the presence of larger size protein complexes in FPs/EG2DM and FPs/EG3DM compared to FPs/EG4DM, which


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is in accordance with the previous results. These observations confirmed the proposed roles of covalent interaction in the self-assembly process.

Figure 3. Initial verification of the formation of protein assemblies. (a) FRET spectra of the FPs mixture (black line) and the co-assemblies with 3 equiv of different linkers (EG2DM, EG3DM, EG4DM). (λex = 405 nm). Inset, photographs of 1) FPs, 2) + EG2DM, 3) + EG3DM and 4) + EG4DM under UV light (365 nm). (b) 12% SDSPAGE analysis of the FPs mixture before and after assembly: lane 1, protein marker; lane 2, FPs; lane 3, FPs/EG2DM; lane 4, FPs/EG3DM; lane 5, FPs/EG4DM. Region I: stacking gel; region II, separation gel.

We further employed fluorescence emission spectroscopy to monitor assembly behavior and evaluate assembly quality. FPs mixture (EBFP2-4C:EGFP-4C = 3:1) was allowed to react with EG3DM in different proportions and then monitored by fluorescence spectroscopy (Figure 4a, b). The discrete FPs in the solution were cross-linked by the added EG3DM to form ordered and closely packed aggregates, that resulted in rapid energy transfer from donor to acceptor. With the ratio of EG3DM/FPs increasing, more FPs participated in the assembly, resulting in the emission intensity at 508 nm enhancing and that at 448 nm rapidly attenuating. However, when above the critical ratio 3:1, an inverse tendency was observed: the excess EG3DM saturated the cysteine on


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the surface of protein, thereby preventing the assembly from proceeding efficiently and resulting in a low energy transfer efficiency. Based on the data above, the ratio of emission intensity at 508 nm and 448 nm (I508/I448) was employed to evaluate the assembly quality (Figure 4c). The value of I508/I448 grew liner with the ratio of EG3DM/FPs increasing and reached the maximum value at the ratio of 3:1. With the further addition of EG3DM, the value of I508/I448 gradually decreased and finally stabilized. Interestingly, the value of I508/I448 is very close when assembling with two or three equiv of EG3DM. And the Cys:maleimide ratio was calculated to be 1:1 when assembling with two equiv of EG3DM, which is in agreement with the theoretical value 1:1. The result also verified the self-assembly process was driven by the covalent interaction from another perspective. In addition, the kinetics behaviors of the self-assembly were also carefully monitored by fluorescence spectroscopy. Figure 4d showed the curve of the I508/I448 of FPs mixture (D:A = 3:1) varying with time, when assembling with 3 equiv of EG3DM.


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Figure 4. Fluorescence spectroscopy studies of the assembly behavior and the kinetics behaviors. (a, b) FRET spectra of the FPs mixture (black line, [EBFP2-4C] = 1 μM) and the co-assemblies in aqueous solutions with various amounts of EG3DM (λex = 405 nm, R = linker:FPs). (c) The ratio of emission intensity at 508 nm and 448 nm (I508/I448) versus the ratio of EG3DM/FPs. Error bars were calculated from at least three independent measurements. (d) The kinetics curve of I508/I448 changing with time when assembled with 3 equiv of EG3DM.

Different from organic dyes, concentration quenching seems to be minimal for FPs owing to their protective β-shell structures. FPs still maintain their high brightness even at a high density (Figure 5a, Figure S12). Figure 5b showed the light-harvesting protein nanosheet formed by ED3DM induced self-assembly, in which the absorbed energy can be transferred within the adjacent donors and was subsequently delivered to the nearby acceptor. The energy transfer from EBFP2-4C to EGFP-4C was confirmed by fluorescence spectroscopy. As shown in Figure 5c, increasing the concentration of the doped acceptor in the co-assembly system decreased the emission intensity of donor at 448 nm while enhanced that of the acceptor at 507 nm when excited at 405 nm. However, no energy transmission was observed in the absence of EG3DM (Figure S13a). On the contrary, while maintaining the concentration of acceptor, the increase in the donor concentration enhanced the emission intensity for both donor and acceptor (Figure 5b). All these results indicated that the donor contributed directly to the acceptor emission and the absorbed energy can transfer efficiently in the artificial light-harvesting system.


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Figure 5. (a) Schematic illustration of the dispersive FPs in solution and the densely packed protein nanosheet with high brightness and conspicuous FRET phenomenon. (b) Schematic illustration of the light-harvesting protein nanosheet in which the absorbed energy can be transferred to acceptor via direct FRET or successive donor-to-donor transfers. (c) FRET spectra of the co-assembly system doped with various amounts of acceptor EGFP-4C. [EBFP2-4C] = 2 μM. (d) FRET spectra of the co-assembly system doped with various amounts of donor EBFP2-4C. [EGFP-4C] = 0.22 μM. (λex = 405 nm)

Further evidence for efficient energy transfer was provided by steady-state and time-resolved fluorescence (Figure 6a, b). Time-resolved fluorescence measurements clearly showed a decrease in the donor fluorescence lifetime (τ) after co-assembling with acceptor, indicating an efficient energy transfer. As expected, simply blending at a low concentration has no influence on the lifetime of the donor. The fluorescence lifetime of the donor EBFP2-4C decreased markedly with the increase of the acceptor-to-donor molar ratio when compared with the lifetime of EBFP2-4C alone (τ = 3.5 ns) (Figure 6b). The shortened lifetime of EBFP2-4C indicated that a non-radiative


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energy transfer occurred from the excited-state EBFP2-4C to the EGFP-4C. Notably, the lifetime of EBFP2-4C in FPs/EG2DM and FPs/EG3DM was very close, which indicated that a slight increase in the distance between adjacent monomers can greatly increase the size of the assembly, but has almost no sacrifice on energy transfer efficiency. The energy-transfer efficiency E was then calculated to evaluate the light-harvesting capability of the assembly system, and the value was calculated to be 33.2% at the ratio of 3:1 for EBFP2-4C to EGFP-4C when induced by EG3DM. Although the efficiency is lower than those based on organic dye molecules, it is more effective than those in conventional peptide-fused donor-acceptor pairs of similar FPs (Table S2).40

Figure 6. (a) Donor fluorescence lifetime decay curves of EBFP2-4C alone (τ = 3.5 ns), the mixture of uncross-linked EBFP2-4C and EGFP-4C (τ = 3.4 ns) and the co-assembly system with EG2DM (τ = 2.2 ns), EG3DM (τ = 2.3 ns) and EG4DM (τ = 2.6 ns). (b) Donor fluorescence decay curves in the absence (black line) and in the presence of different concentrations of acceptor. (λ = 375 nm, D:A = donor:acceptor)

To improve light-harvesting efficiency, the quenching problem should be minimized first. Our work is designed inspired by the glowing jellyfish. In our work, we succeeded in minimizing energy quenching without relying on any templates, which provided an idea for avoiding the energy loss caused by aggregation quenching. Compared with those reported, we developed a


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simple way to build a light-harvesting system without time-consuming synthesis process. More importantly, we believe that this bio-optimistic structural feature of fluorescent proteins can provide insights into the optimization of the optoelectronic properties of dye molecules. It has been shown that sequestering dyes in protective host molecules may maintain or enhance the optoelectronic properties in the solution or even solid state.41,42 This is very meaningful for the further development of artificial light-harvesting systems. CONCLUSION In conclusion, we have successfully constructed a template-free, highly ordered 2D lightharvesting system completely based on FPs. The excellent anti-self-quenching property of fluorescent proteins provided the possibility for constructing such a template-free light-harvesting system. Covalent-induced protein self-assembly provided a facile means for organizing a large number of FPs in an orderly manner to form long-range order, monolayered protein nanosheets, in which energy transfer efficiently with almost no self-quenching. In this work, the covalentinduced self-assembly process and energy transfer process have been studied in detail. What is more, we demonstrated the profound influence of the non-negligible secondary interactions between protein surfaces on the construction of light-harvesting system where even a minor change in the length of the inducing linker exhibited a significant effect on the size of the assemblies. By using EG3DM as linker, a micron-sized light-harvesting system with satisfactory energy transfer efficiency was finally obtained. Not limited to this work, the special core-shell structure of FPs may be expected to direct the optimization of organic dyes, such as enhancing the fluorescence intensity and fluorescence stability through cladding. Following this mindset, more efficient and more creative light-harvesting systems may be constructed in the future.


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EXPERIMENTAL SECTION Construction, Expression and Purification of EGFP and EBFP2 Variants. As a derivative of EGFP, EBFP2 share almost the same structure and amino acid sequence with EGFP. Therefore, we performed the same site-directed mutagenesis for the two proteins. Depending on the structural information of fluorescent protein, four sites with approximately C4 symmetry on the side of the beta-barrel were screened and then mutated into cysteine. The mutant plasmids pET22b-EGFP-4C (E32C-C48G-E124C-K166C-Q204C) and pET-30a-EBFP2-4C (E32C-C48GE124C-K166C-Q204C) were designed and subsequently synthesized directly by the Sangon Biotech. The resulting plasmids were confirmed by genetic sequencing and then transformed into BL21 E. coli cells for proteins expression. The BL21 E. coli strains containing the mutant EGFP and EBFP2 genes were cultured in 1 L LB liquid medium containing 100 μg/ml ampicillin and kanamycin, respectively, with rotary shaking at 37 °C until OD600 reached 0.6. Isopropyl β-D-1thiogalactopyranoside (IPTG) was added at a final concentration of 1.0 mM to induce protein expression. After 12 hours of induction at 22 °C, the induced cells were harvested by centrifugation at 8000 rpm for 15 min and sonicated in 20 mM Tris-HCl, pH = 7.9, containing 500 mM NaCl for subsequent Ni-NTA purification. The protein was then purified by the Ni-NAT purification system with a gradient of imidazole from 30 mM to 500 mM and the target protein was eluted with 300 mM imidazole. Excessive imidazole and NaCl were removed by dialysis. The purity of the two proteins was characterized by SDS-PAGE gel electrophoresis (Figure S5a) and the molecular weights were performed by MALDI-TOF mass spectrometry (Figure S5b, c). Self-Assembly of FPs Nanosheets. EBFP2-4C and EGFP-4C were dissolved in Tris-HCl buffer (10 mM Tris-HCl, 20 mM NaCl, pH = 7.4) respectively and mixed at a ratio of 3:1 to a final protein concentration of 12 μM. Cross-linker was dissolved in pure water at a concentration of


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0.1 mM as a stock solution. The aqueous solution of linker (180 μl, 0.1 mM) was added dropwise into a 500 μl sample of protein solution. The mixtures were stirred gently for 10 min and further incubated overnight at 4 ºC. Purification of Protein Assemblies. The assembled samples were injected onto a SEC 200Å column and run at 0.4 ml/min with 20 mM sodium phosphate buffer (pH = 7.0) as elution buffer. Protein elution was monitored at 280 nm. Large-scaled assemblies (retention time 6-7 min) are separated for subsequent morphology characterization. Atomic Force Microscopy. Atomic force microscopy (AFM) measurements were performed using a NanoScope Multimode AFM with a Nanoscope III controller (Veeco Metrology, Santa Barbara, CA) in tapping mode with a SiN4 probe. A 10 µl aliquot of sample was dispersed on freshly cleaved silicon wafer, incubated for 5 min. The excess solution was removed by pipette and then the samples were dried in air. Transmission Electron Microscopy. Transmission electron microscopy (TEM) measurements were performed using a JEM-2100F transmission electron microscope with 200 kV accelerating voltage. 10 μl sample was adsorbed to carbon-coated copper grids for 8 min. The excess solution was drawn away by pipette. The samples were then negatively stained with 2% uranyl acetate aqueous solution for 2 min and then the excess stain was removed. The samples were dried in air before measurement. Dynamic Light Scattering. Dynamic light scattering (DLS) experiments were carried out with Malvern Instrument Zetasizer Nano ZS at 25 ºC to study the hydrodynamic diameters of the assemblies formed with three different linkers (Figure S8a). EBFP2-4C and EGFP-4C were dissolved in Tris-HCl buffer (10 mM Tris-HCl, 20 mM NaCl, pH = 7.4) respectively, mixed at a


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ratio of 3:1 to a final protein concentration of 12 μM and reacted with three equiv of linkers. The samples incubated overnight were diluted with filtered water for testing. To study the protein self-assembly process, the hydrodynamic diameters of the assemblies were also recorded in different time scales (Figure S8b). Fluorescence Spectroscopy. Fluorescence spectrometry was performed using RF-5301PC Spectrofluorophotometer (Shimadzu, Japan). To avoid concentration-caused self-quenching, the high concentration of protein sample was first diluted with pure water to an appropriate concentration and then loaded into a 4 mm quartz cell for the measurement of spectra. Fluorescence emission spectra were recorded from 300 nm to 600 nm with an excitation wavelength of 405 nm, 3 nm excitation bandwidth, 3 nm emission bandwidth and the results were collected by averaging at least three measurements. ASSOCIATED CONTENT Supporting Information Available: Details of the synthesis procedures and characterizations (1H NMR, ESI-MS) of linkers; Design, expression and characterization (SDS-PAGE, MALDITOF) of EGFP and EBFP2 variants; Morphology characterization of EG3DM and EG4DM induced FPs self-assemblies; Control experiments of the FRET and calculation of energy transfer efficiency; Comparison of the efficiency for light-harvesting and FRET between the assembly and EGFP/4EBFP2 and 4EGFP/EBFP2; Analysis of the effect of the sheet size on FRET. These materials are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author


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*Junqiu Liu. State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China; E-mail: [email protected] ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21420102007, 21574056 and 91527302). REFERENCES 1. Peng, H. Q.; Niu, L. Y.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.; Yang, Q. Z. Biological Applications of Supramolecular Assemblies Designed for Excitation Energy Transfer. Chem. Rev. 2015, 115, 7502–7542. 2. Wasielewski, M. R. Self-Assembly Strategies for Integrating Light Harvesting and Charge Separation in Artificial Photosynthetic Systems. Acc. Chem. Res. 2009, 42, 1910–1921. 3. Ishida, Y.; Shimada, T.; Masui, D.; Tachibana, H.; Inoue, H.; Takagi, S. Efficient Excited Energy Transfer Reaction in Clay/Porphyrin Complex toward an Artificial Light-Harvesting System. J. Am. Chem. Soc. 2011, 133, 14280–14286. 4. Lee, C. Y.; Farha, O. K.; Hong, B. J.; Sarjeant, A. A.; Nguyen, S. T.; Hupp, J. T. LightHarvesting Metal-Organic Frameworks (MOFs): Efficient Strut-to-Strut Energy Transfer in Bodipy and Porphyrin-Based MOFs. J. Am. Chem. Soc. 2011, 133, 15858–15861. 5. Zeng, Y.; Li, Y. Y.; Li, M.; Yang, G. Q.; Li, Y. Enhancement of Energy Utilization in LightHarvesting Dendrimers by the Pseudorotaxane Formation at Periphery. J. Am. Chem. Soc. 2009, 131, 9100–9106.


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Template-Free Construction of Highly Ordered Monolayered

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