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Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme–Heme Exciton Coupling Koji Oohora, Nishiki Fujimaki, Ryota Kajihara, Hiroki Watanabe, Takayuki Uchihashi, and Takashi Hayashi J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b06690 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 1, 2018
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Journal of the American Chemical Society
Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme–Heme Exciton Coupling Koji Oohora,*†‡§ Nishiki Fujimaki,† Ryota Kajihara,† Hiroki Watanabe,‖ Takayuki Uchihashi,*‖ Takashi Hayashi*† †
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan § PRESTO, Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Japan ‡
‖Department
of Physics, Nagoya University, Nagoya, 464-8602, Japan
Supporting Information Placeholder ABSTRACT: A supramolecular assembly of units of cytochrome b562 with externally attached heme having intermolecular linkages formed via the heme–heme pocket interaction was investigated in an effort to construct a welldefined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome b562 provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of observation of a split-type Cotton effect by heme–heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images and mutation experiments, reveal that the assembly has a periodic helical structure with 3-nm pitches, suggesting the formation of the assembled structure is driven not only by the heme–heme pocket interaction but also by additional secondary hydrogen-bonding and/or electrostatic interactions at the protein interfaces of the assembly. In nature, combinations of multiple interactions provide the well-defined supramolecular assembly structures such as the helical duplex of DNA, as well as fibrous, ringshaped or cage-like protein assemblies.1 These elegant and systematic structures have encouraged chemists and biologists to generate artificial protein assemblies with various structures including rings,2 2D sheets,3 tubes4 and cages.5 However, examples of generation of well-defined structures remain limited and few systems have been evaluated in solution. Helical structures consisting of small chromophore assemblies are known to produce a characteristic split-type Cotton effect, which is a helpful indicator in evaluation of the structure by examination of circular dichroism (CD) spectra of chromophore assemblies in solution.6 In this context, we have focused on the construction of protein assemblies based on hemoproteins with a cofactor, protoporphyrin IX iron complex (heme), as a characteristic chromophore.7 Introduction of a covalently attached heme molecule onto the apoprotein surface will trigger formation of the supramolecular hemoprotein assembly via
the heme–heme pocket interaction as a driving force. Based on this concept, our efforts demonstrated one,8 two9 and three dimensional10 structures of assembled hemoproteins. However, in our previous work, we have only been able to generate the flexible structures. Thus, we concluded that an additional set of secondary interactions in addition to the direct heme–heme pocket interaction will be required in order to generate a more rigid and well-defined structure. Here, we provide a first report of a new artificial system based on a periodic and relatively rigid hemoprotein assembly which exhibits exciton coupling between the chromophores (Figure 1).
Figure 1. (a) NMR structure of cytochrome b562 (Cyt b562, PDB ID: 1QPU). (b) Surface potential map of Cyt b562 generated by PyMOL. Heme and N80 moieties are highlighted in yellow. (c) Schematic representation of the process for generating the supramolecular assembly of Cyt b562 mutants.
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Figure 2. (a) SEC traces of 1-N80C (red), 1-H63C (blue) and authentic samples including monomeric Cyt b562 (black). (b) UV-vis absorption spectra of 1-N80C assembly (red) and 1-H63C assembly (blue). (c) CD spectra of the 1N80C assembly (red), the 1-H63C assembly (blue) and monomeric Cyt b562 (black). (d) CD spectra of the 1-N80C assembly at various salt concentrations. (e) CD spectra of the 1-N80C assembly under the various pH conditions. Cytochrome b562 (Cyt b562), one of the smallest known electron transfer hemoproteins, has been employed as a hemoprotein building block in a supramolecular assembly due to its solubility and characteristic absorption derived from the hexa-coordinated low spin heme. Previously, we selected the residue 63 as the site for attachment of heme to the Cyt b562 surface and prepared the H63C mutant8,9 because this residue is located on the opposite side of the heme-binding site as indicated by the NMR structure (Figure 1a).11 In the present work, the residue 80 was newly chosen as an alternative heme attachment site due to its complementarily electrostatic potentials with respect to the Asn80 residue vs. other residues near the exit of the hemebinding site. In contrast, the predominately negativelycharged residues near introduced Cys63 residue might cause repulsive interactions with other residues near the exit of the heme-binding site at the protein interface (Figure 1b). Favorable secondary interactions such as electrostatic and hydrogen bonding interactions were expected to occur at the protein interface between the surroundings of the heme-binding site and the heme attachment site of an adjacent Cyt b562 molecule. To maximize the effect of the secondary interaction at the protein interface, we employed
maleimide-tethered heme 1 with the short linker between heme and maleimide moieties. The N80C mutant prepared using the conventional E. coli expression system was modified by attaching maleimide-tethered heme 1 to yield the monomer unit, 1-N80C (Figure 1c). The modified protein is characterized by LCMS: found m/z = 1251.70, calcd m/z = 1251.75 (z = 10+) (Figure S1). Analytical size exclusion chromatography (SEC) for 1-N80C shows a much larger assembly than that observed for 1-H63C under the same concentration of monomeric protein and the peak top indicates approximately a 10-mer size (Figure 2a).12 The UV-vis spectrum of 1N80C assembly measured at neutral pH is consistent with the previously reported protein assembly, 1-H63C assembly (Figure 2b). The spectrum is typical of hexacoordinated low spin species, indicating successful incorporation of the attached-heme on the protein surface into the heme-binding site of an adjacent protein molecule. These data demonstrate the successful formation of the 1N80C assembly via the heme–heme pocket interaction. In circular dichroism (CD) spectra of the Soret band region (wavelengths between 390-480 nm) (Figure 2c), it is seen that the wild type protein and the 1-H63C assembly provide only a negative Cotton effect, which is induced by exciton coupling interaction between heme and amino acid residues at the heme-binding site.13 Interestingly, a splittype Cotton effect is seen in the CD spectrum of the 1N80C assembly, although the UV-vis spectrum of 1-N80C assembly is similar to that of Cyt b562 (vide supra). Splittype Cotton effects are often observed in stericallyconstrained dimers of chromopohores.14 Thus, it appears that the split-type Cotton effect observed in the CD spectrum of 1-N80C assembly is arises from an exciton coupling interaction derived from the sterically-constrained heme chromophores in the assembled protein structure. This finding provides the evidence for a rigid structure supported by the expected secondary protein–protein interaction. The existence of the secondary interaction causes a peak shift to an earlier elution volume in the SEC profile (vide supra). To confirm the character of this interaction, we measured CD spectra under several conditions. First, the salt strength was increased by addition of NaCl to 1 M as a final concentration (Figure 2d). However, no change in CD spectrum was detected, suggesting that the secondary interaction is not simply electrostatic in nature. Second, the pH values were decreased gradually (Figure 2e). In contrast to the salt strength experiment, decreasing the pH caused drastic changes in the CD spectrum. At pH 3.0, only a negative Cotton effect was observed for the 1-N80C assembly and the Cotton effect disappeared at pH 1.8. The set of secondary interactions appears to be broken at pH 3.0 and the heme–heme pocket interaction is ineffective at pH 1.8, which is consistent with observations made previously.8,15 These results indicate that in addition to general electrostatic interactions, a strong-salt bridge and/or a hydrogen-bonding network constrain the structure of the assembly.
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Journal of the American Chemical Society 66 fibrils, Figure 3c) was statistically analyzed by the Gauss-fitting and the value was determined to be 2.9 ± 0.02 nm. This deviation is likely within the measurement error under these conditions. Thus, these AFM results experimentally demonstrate the periodicity of the structure derived from the rigid protein assembly, as be suggested by CD spectrum. Figure 3. (a) Representative high-speed AFM image of the 1-N80C assembly. (b) Height profile along a red line in the image (a). (c) Histogram of pitches from the height profiles.
Figure 4. (a) Optimized structure provided by MD simulation of the dimer model of the 1-N80C assembly. Cyan and purple proteins are apo-Cyt b562 and 1-N80C with native heme, respectively. (b) The plots of important structural factors, heme–heme dihedral angles (red) and Fe–Fe distances (green), of the dimer model against the simulation. (c) Simulated CD spectrum based on the dimer model (purple) and the experimental CD spectrum (orange). (d) and (e) Schematic representation of interacting amino acid residues in the dimer model. (f) CD spectra of 1-N80C, 1N80C/R98A and the 1-N80C/D73A assemblies. High-speed atomic force microscopy (AFM) reveals the shape of the 1-N80C assembly weakly immobilized on poly-lysine-coated mica under solution conditions. The fibrous structures were observed with good reproducibility (See supporting movies S1 and S2). The lengths of the structures were found to be between about a dozen nanometers to the sub-micrometer range, which indicates polymers ranging from 5-mers to 50-mers. Figures 3a and 3b show the representative AFM image and the height profile along the red line in the image, respectively. Interestingly, the periodicity of the tops can be clearly seen in the profile. A histogram of the pitches of the tops (n = 655 for
Figure 5. (a) MD-optimized structure of the nonamer model of the 1-N80C assembly. (b) The model of AFM simulation: the probe is represented by the yellow triangle and the MD-optimized nonamer was employed. (c) Simulated AFM image. (d) The height profile along a red line in the image (c). To estimate the precise morphology and atomic scale structure, molecular dynamics (MD) simulation was performed using a dimer model designed as the complex of apo-Cyt b562 and 1-N80C with native heme inserted into the heme pocket (Figure 4a). Four MD simulations of the dimer models with independent initial structures provided different structures after 225-ns simulations (Figures S2– S5). Heme–heme dihedral angles and Fe–Fe distances which are important parameters for the heme–heme exciton coupling were monitored in the MD simulations (Figure 4b). The accuracy of each MD-optimized structure is evaluated in a comparison between the simulated CD spectrum using an empirical exciton coupling method based on the obtained coordinate data of the chromophore structures and the observed CD spectrum.14a,b One of the four independent simulations reproduced the experimental split-type Cotton effect to a significant degree (Figure 4c). The MDoptimized structure validated by the simulated CD spectrum suggests the presence of a hydrogen bonding network including Arg98, Arg106, Asp73’ and Asp74’ (Figures 4d and e) as a secondary interaction. To investigate this possibility, two alanine mutants of N80C, N80C/D73A and N80C/R98A, were prepared and linked with 1 to form the supramolecular assemblies, 1-N80C/D73A and 1N80C/R98A assemblies, respectively. Surprisingly, the characteristic split-type Cotton effects disappeared and only negative Cotton effects were observed in the CD spectra of 1-N80C/D73A and 1-N80C/R98A assemblies (Figure 4f). The CD spectrum of the 1-N80C/D73A assembly is the most consistent with that of Cyt b562.16 These dramatic differences in CD spectra provide a strong indication that the present MD simulation is accurate. Furthermore, the nonamer was constructed based on this dimer model and optimized in a 10-ns MD simulation (Figure 5a). The obtained protein structure has an apparent pseudo-three-fold
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helical axis. An AFM simulation was carried out based on this nonamer structure on the flat substrate using a probe with a 0.5 nm tip radius (Figure 5b). The pitch of the tops in the simulated image is approximately 3 nm, which is consistent with the value determined experimentally by AFM (vide supra). Thus, the multiple experimental results provide quantitative support for the accuracy of the MDsimulated structure In conclusion, selection of an appropriate position for attachment of heme to the protein surface is important in order to obtain a supramolecular hemoprotein assembly with a periodic structure via a heme–heme pocket interaction with an additional secondary interaction, as indicated by the characteristic split-type Cotton effect and wellsimulated MD structure. To the best of our knowledge, the heme–heme exciton coupling was first observed in a series of artificial assembling systems of hemoproteins.4a, 5c,d, 7-10 In terms of this feature observed in the normal solution state, the structurally well-defined assembly in the present system is clearly distinguished from other artificial hemoprotein assemblies with the fine structures formed under the specific crystallization conditions.7a The provision of a set of secondary interactions via hydrogen-bonding and/or electrostatic interactions at the protein interfaces is important to effectively control structural properties such as rigidity and configuration of the hemoprotein assemblies. It is expected that further design and construction of hemoprotein assemblies will contribute to development of smart biomaterials such as allosteric small molecule carriers and electronic wires.
ASSOCIATED CONTENT Supporting Information. Experimental details and characterization data are available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author
[email protected] [email protected] [email protected] ACKNOWLEDGMENT We appreciate Prof. Ulrich Schwaneberg and Dr. Marco Bocola (RWTH Aachen Univ.) for the discussion about the MD simulations. This work was supported by Grants-in-Aid for Scientific Research provided by JSPS KAKENHI Grant Numbers JP15H05804, JP15H00873, JP16K14036, JP16H06045, JP16H00837, JP16H00758 and JP18H04512. We appreciate support from JST PRESTO (JPMJPR15S2) and SICORP.
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