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The Crystal Structure of a Maxi-/Mini-Ferritin Chimera Reveals Guiding Principles for the Assembly of Protein Cages Thomas Cornell, Yogesh Srivastava, Ralf Jauch, Rongli Fan, and Brendan Orner Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00312 • Publication Date (Web): 06 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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The Crystal Structure of a Maxi-/Mini-Ferritin Chimera Reveals Guiding Principles for the Assembly of Protein Cages Thomas A. Cornell,†,‡,∥ Yogesh Srivastava, § Ralf Jauch,ǂ,§ Rongli Fan,‡ Brendan P. Orner†,‡,* †

Department of Chemistry, King’s College London, UK.

‡

Division of Chemistry and Biological Chemistry, Nanyang Technological University,

Singapore. ǂ

Genome Institute of Singapore, Singapore.

§

Genome Regulation Laboratory, Guangzhou Institutes of Biomedicine and Health, Chinese

Academy of Sciences, Guangzhou, China.

KEYWORDS Ferritin, Bacterioferritin (Bfr), DNA binding protein from starved cells (Dps), Ehelix, Quaternary structure, Self-assembly, Protein-protein interactions, Protein cage.

ABSTRACT. Cage proteins assemble into nanoscale structures with large central cavities. They play roles including virus capsids and chaperones, and have been applied to drug delivery and nanomaterials. Furthermore, protein cages have been used as model systems to understand and

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design protein quaternary structure. Ferritins are ubiquitous protein cages that manage iron homeostasis and oxidative damage.

Two ferritin subfamilies have strongly similar tertiary

structure yet distinct quaternary structure: maxi-ferritins normally assemble into 24-meric, octahedral cages with C-terminal E-helices centered around four-fold symmetry axes and miniferritins are 12-meric, tetrahedral cages with three-fold axes defined by C-termini lacking Edomains. To understand the role E-domains play in ferritin quaternary structure, we previously designed a chimera of a maxi-ferritin E-domain fused to the C-terminus of a mini-ferritin. The chimera is a 12-mer cage midway in size between that of the maxi- and mini-ferritin. The research described herein, sets out to understand a) whether the increase in size over a typical mini-ferritin is due to a frozen state where the E-domain is flipped out of the cage and b) whether the symmetrical preference of the E-domain in the maxi-ferritin (four-fold axis) overrules the Cterminal preference in the mini-ferritin (three-fold axis). With a 1.99 Å resolution crystal structure, we determined that the chimera assembles into a tetrahedral cage nearly superimposable with the parent mini-ferritin, and that the E-domains are flipped external to the cage at the three-fold symmetry axes.

INTRODUCTION. Members of the protein cage structural class fold into monomers that assemble into large, hollow architectures often on the nano-scale.1, 2 These proteins have cellular functions as wide reaching as protein folding,3, 4 metabolic enzymology,5 and atom and molecule sequestration and transport.6 Moreover, they have been of great interest to structural biology due to their complex quaternary structure, often governed by protein-protein interactions, and have recently been the focus of protein design and engineering studies.7,

8, 9,10, 11

Because of their

unique structure12, protein cages have been applied to the generation of nanomaterials,13, delivery,16, 17 and catalysis.18


14,15

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Ferritin proteins are well-characterized protein cages and have been the subject of much research due to their ubiquity across species,19 their essential function,20 and their wellestablished structures with relatively low complexity compared to other protein cages. Ferritins function as a cellular iron store through mineralization within the protein central cavity.6, 7, 21,22 This results in sequestration of iron to lessen of the risk of oxidative Fenton chemistry while assisting iron homeostasis. We have used ferritins as model systems to understand the structural energetics of protein cages. 1, 8, 9, 23, 24, 25 Although the ferritins share broad sequence identity, their monomer tertiary structures26 based around four-helix bundles are strikingly homologous27. This structural similarity is carried through to the quaternary structure with only a few distinct subfamilies. Maxi-ferritins are assembled from twenty-four monomer subunits and form nanocages with outer and inner diameters around 12 and 7 nm respectively and octahedral symmetry.28 Mini-ferritins are assembled from twelve monomers into a cage with tetrahedral symmetry. One typical miniferritin, E. coli DNA-binding protein from starved cells (Dps), forms cages with outer and inner diameters of 9 nm and 4.5 nm respectively.29 The four-helix bundle monomers of both the mini- and maxi-ferritins are thought to assemble into cages through a dimer intermediate which is assumed to be structurally similar to the antiparallel dimer found at the two-fold axis of symmetry in both cages.1, 30, 31 In the maxiferritin octahedral cage, the three-fold axis of symmetry is located at a trimeric interface near the N-termini of the monomers and the four-fold axis is at a tetrameric interface centered around the C-termini.32 The mini-ferritin tetrahedral cage is defined by a three-fold axis centered at the monomer N-termini (the “maxi-ferritin-like axis”) and another centered at the C-termini (the “mini-ferritin-like axis”).30 While the monomers of the mini- and maxi-ferritins are notably


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similar four helix bundles, the tertiary structures differ by the presence of additional fifth helices that are only two or three helical turns. Maxi-ferritins tend to have an additional helix at the Cterminus of the monomer (the “E-helix”), near the four-fold symmetry axis in the cage, and miniferritins often have a fifth helix in the loop between the second and third helix of the bundle (the “BC-helix”), and it is oriented across the two-fold symmetry axis. We have been interested in understanding the assembly of both the bacterioferritin from E. coli (Bfr), a maxi-ferritin, and E. coli Dps, a mini-ferritin. We focus on these proteins because of their straightforward expression in E. coli and the fact that the maxi-ferritin is a homooligomer, unlike mammalian ferritins which thus have more complicated assembly. We have discovered “hot spot” residues that disproportionately influence stability across protein-protein interfaces using “alanine shaving” mutagenesis.33, 7 In addition, we have rationally re-engineered protein-protein interfaces to stabilize and more efficiently assemble the cage.8,

9

Recently we

have developed a medium throughput screen for assembly at key symmetry interfaces in the cage25, 34 that we have expanded to select cages with novel properties from protein libraries.35 Moreover, through the design of deletion, swapping, and chimeric Bfr and Dps mutants, we have explored the role of the maxi-ferritin E-helix and the mini-ferritin BC-helix in structure formation.23 Through the course of that study, we determined that the E-helix seemed to play a larger role in cage structure than does the BC-helix. In addition, using size exclusion chromatography (SEC) (Fig. S6), dynamic light scattering, and transmission electron microscopy (Fig. S10), we found that that the chimeric protein, Dps+E, which was designed by fusing the maxi-ferritin E-domain to the mini-ferritin C-terminus, assembled into a discrete cage of unusual size (consistent with a 16-mer). However, sedimentation equilibrium confirmed that it was 12-


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meric, consistent with the oligomerization state of the parent mini-ferritin, but suggesting that Dps+E may be structurally and conformationally distinct from both Dps and Bfr. Although we had established that Dps+E is larger than Dps, but smaller than Bfr, and was made up of twelve monomers like Dps, it was unclear how this actually manifested in the structure of Dps+E. Based on this data, we had two initial questions. First, because the E- helix in Bfr is positioned at the four-fold axis of symmetry in octahedral Bfr, but Dps is tetrahedral, without four-fold symmetry, we were curious if the four-fold or three-fold symmetric quaternary structure would prevail. In other words, would the structural preference of the Dps monomer or the Bfr E-helix control the geometry of the cage? (Figure 1) The second question involved an older paper from the ferritin literature36. This report speculated that the E-helix, which is normally pointing into the protein cage cavity in octahedral maxi-ferritins, could “flop” in and “flip” out of the nanocage in a dynamic manner. We therefore were curious whether Dps+E was frozen in a “flipped out” state, thus making it appear larger while maintaining the 12-meric stoichiometry. (Figure 1) A good way to obtain answers to these questions is to obtain the x-ray crystal structure of Dps+E. A crystal structure should be able to definitively answer these questions and provide high resolution information about key protein-protein interactions and whether they have been altered from those in Bfr or Dps. In addition, the crystal structure could provide fundamental insight into the structural energetics of protein cages in general, and this insight could help to engineer similar or even distinct synthetic assemblies in the future.


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A)

B)

C)

Does the mini-ferritin control symmetry in the chimera?

WT mini-ferritin 3-fold axis (No E-domain)

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Is the chimera frozen in the “ ipped out state”? E-Domain Flip-Flop

Does the E-domain control symmetry in the chimera?

WT maxi-ferritin 4-fold axis (with E-domain)

Figure 1. (A) Crystals structures of DNA binding protein from starved cells (Dps) from E. coli (PDB:1DPS), which is a mini-ferritin and has no E-domain, viewed down the three-fold symmetry axis formed by C-terminal interactions of three monomers highlighted in dark grey, and the maxi-ferritin, E. coli bacterioferritin (Bfr) (pdb:1BFR), viewed down the four-fold symmetry axis formed around the C-terminal E-helices for four monomers (highlighted in dark grey). The study presented herein builds upon our previous design of Dps+E which is a chimeric fusion of the mini-ferritin with the Bfr E-domain.23 (C) The ability of the C- terminal E-helix to flip in and out of the cage is the foundation of the “Flip-Flop” hypothesis of maxi- ferritin assembly.36 We set out to determine if Dps+E is frozen in the “flipped out” state. and (B) if the symmetry preference of the Bfr E-domain or the Dps cage prevails in Dps+E.

METHODS AND MATERIALS.


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Plasmid construction, expression, and protein purification are described in the Supporting Information. Crystallization  Once the protein was purified, the buffer of the resulting Dps+E solution was exchanged (10 mM NaCl, 10 mM HEPES, pH 7) and the protein was concentrated (15 mg/ml) for screening of crystallization conditions. Initial high throughput screens were set up using robotics in 96-well plates with a sitting drop technique (JCSG+ (Qiagen), Classic suite (Qiagen), Morpheus suite (Molecular Dynamics), PEG suite I (Qiagen) and PEG suite II (Qiagen)). Further crystal screening took place in 15-well plates using the hanging drop vapor diffusion technique (see Supporting Information S8 for all crystal screening conditions). The crystal for the protein structure described herein was grown using 0.2 M lithium sulphate, 18% PEG 1000, pH 4.2. Detectable crystals appeared after 3 - 4 days at 19 °C. Data collection, processing, and structure solution A 1.99 Å data set was obtained from the National Synchrotron Light source (NSLS) at Brookhaven National Labs. The data was integrated, scaled and merged using HKL2000.37 38 Phaser (CCP4) was used for molecular replacement experiments, using twelve copies of a polyalanine monomer of wild type Dps from E.coli (1DPS) obtained using Chainsaw.39 This structure has around 85% similarity with the expected new structure of DPS+E. Parrot40 was used to improve the phasing from the molecular replacement. The model was built manually in COOT

41

using 2Fo-Fo and Fo-Fc maps. The refinement was done using Rermac5

42

from CCP4

applying NCS restraints with default internal weighting term (default = 1.0) and an external weighting term of 0.3. Translation/Liberation/Screw (TLS) refinement was conducted towards


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the final steps of the refinement.43 Each monomer of the cage was assigned its own TLS group, giving 12 TLS groups in total. Final validation was performed using Phenix Validate.44 Calculation of RMSD The coordinates for the crystal structures of both wild type Dps (PDB:1DPS29) and Dps+E were aligned using Chimera45 with the Matchmaker tool, which uses a NeedlemanWunsch alignment algorithm. This tool focuses on aligning alpha carbon atoms after sequence alignments have been performed. From this overlay, least square fitting was used to calculate RMSD (see Supporting information S9 for full list of RMSD values per residue).

RESULTS AND DISCUSSION Dps+E was expressed and purified and characterization showed the presence of a nanocage consistent with that previously reported29 (see Supporting Information S4, S5, and S6 for SDS-PAGE purification analysis, mass spec, and size exclusion chromatography (SEC) data). Purified Dps+E was subjected to high throughput robotic-assisted crystal screening (see Supporting Information S7 for full range of conditions screened) using the sitting drop method to establish first generation conditions. Manual second round screens used hanging drop. Crystals grown in 18% PEG1000, 0.2 M lithium sulphate, 100 mM sodium acetate trihydrate, pH 4.2 (see Supporting Information S8 for crystal microscopy images) provided the highest resolution (2.0 Å) diffraction data when subjected to synchrotron radiation and were used for structure elucidation. As it was already determined through sedimentation equilibrium that Dps+E contained twelve monomers,23 molecular replacement was performed assigning 12-copies of a polyalanine search model (prepared with Chainsaw39) in the unit cell. After molecular replacement


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(Phaser38), the structure was built using Coot41 and refined with Refmac42 and validated with Phenix.44 The final model comprises a 2.0 Å crystal structure of the assembled Dps+E cage. (Table 1). Dps+E Values for the highest resolution shell in parentheses. Method – Vapour diffusion, hanging drop pH – 4.2 Temperature – 292.0 K Method – 18% PEG 1000, 0.1 M sodium acetate trihydrate, 0.2 M Lithium sulfate a

Data Collection Space group Wavelength Cell dimensions

P 21 21 21 1.075 Å A = 103.14 Å B = 104.72Å C = 207.76Å α = 90.0 ⁰ β = 90.0 ⁰ γ = 90.0 ⁰

Resolution (Å) Rmerge (%) Completeness (%) I/σI Redundancy

50.00 – 2.00 (2.07-2.00) 6.9 (51.7) 93.5 (98.5) 320.3/11.5 12.8 (11.9)

Refinement Resolution (Å) No. Reflections used No. Reflections in test set Rwork/Rfree (%)

46.8 – 1.99 135428 7156 17.9/20.8

No. Atoms Protein Water ClNon hydrogen atoms

16563 1781 2 16563


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Mean isotropic B value R.M.S deviations from ideal Bond lengths (Å) Bond angles (◦) Ramachandran analysis (%) Favored Additionally allowed Disallowed

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32.98 0.018 1.786 98.70 1.30 0

Table 1. Data collection and refinement statistics for the crystal structure of Dps+E. Inspection of the Dps+E crystal structure shows that the protein assembles into a protein cage with tetrahedral symmetry that is very similar to that of the parent protein, Dps (Figure 2a). In fact, the global Route Mean Square Deviation (RMSD) for the overlay of the regions of the monomers with identical sequences was 0.185 Å, demonstrating strong similarity between the two structures (for individual amino acid RMSD comparisons see Supporting Information S9). Thus, the question posed at the outset of this study regarding whether the symmetry requirements of the parent cage (three-fold symmetry at the C-terminus) or those of the E-domain (four-fold symmetry at the C-terminus) would prevail, can be definitively answered. Our structure demonstrates that the symmetry requirements of the parent protein, Dps, overcome those of the residues from the Bfr E-helix. The chimeric protein, Dps+E, clearly assembles into a protein nanocage that is tetrahedral and not octahedral (or some other structure with four-fold symmetry), and the residues from the E-domain are positioned at a three-fold axis of symmetry, not a four-fold symmetry axis.


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Figure 2. A) Comparison of the crystal structures for wild type Dps (PDB:1DPS29, white) and for the DPS+E chimeric protein (grey). Comparison of monomers (top) and assembled cages (bottom) with a single monomer in the assembled DPS+E in dark grey. Region in black dashed box shows the position of the C-terminus. Image created with Chimera45 with overlay generated with the ‘match’ function. B) Electron density map 2mFo-DFc (σ =1) with constructed structure


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for the protein Dps+E highlighting the fourth residue of the fused E-domain of Bfr. Note the fall off in electron density beyond this residue. Image was generated with Chimera 1.11 software45. The second question this study initially posed can also be definitively answered, however in a slightly less satisfying way as our structure of Dps+E displayed only limited electron density in the C-terminal E-domain. Although several residues from the fusion could be modeled, the structure of the complete domain could not be reliably determined due to structural disorder. However, enough data could be obtained to confirm that these residues are pointing external to the assembled protein and, thus, the domain is “flipped out” of the cage (Figure 2b). In addition, taken together, these results explain how the Dps+E nanocage can be made up of only twelve monomers, as confirmed by sedimentation equilibrium, but appears larger than Dps in SEC and DLS23. This increase in hydrodynamic radius is a result of the E-domains of the monomers flipped to the outside of the assembled, 12-mer cage. While the aims set out in this study, (to obtain the Dps+E crystal structure, confirm the overall symmetry, determine the position of the E-domains with respect to the nanocage, and understand why the protein is larger in size than Dps) were all fulfilled, we were not able to observe electron density for the entire E-domain. Therefore it is not possible to determine if there are any interactions between the E-domains of adjacent monomers and the degree to which they are unstructured. Yet, as the domain is structurally disordered, any potential interaction is likely transient or structurally irregular in the solid state because more highly defined electron density would be expected if these interactions were more structurally stable. With these termini projecting away from the nanocage on the three-fold axes of symmetry, it was thought that they might have eventual engineering utility as “handles” for applications like drug delivery or supraassembled materials. Therefore we intend to pursue high resolution electron microscopy studies


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to understand the relative positions of these domains in non-crystalline conditions. In addition, it should be emphasized that conditions in the crystalline state are not solution conditions where additional conformations can be explored and assembly dynamics may play a role. Thus we are interested in performing direct, solution-based techniques, perhaps modifications of our bisarsinic fluorescent probe-based assembly assay25, to confirm the position of the E-domains in DPS+E. Although the complete structure of the E-domain could not be determined, the fact that the chimeric protein favors the assembly state of the monomer (12-meric tetrahedron) over that of the E-domain (24-meric octahedron) to the extent that the E-domain is pushed out of the cage and loses some degree of structure, helps direct further questions regarding the formation of higher-order, closed protein structure. It should be remembered that structural stability is dependent upon both enthalpic and entropic terms, and, in this system, the latter can be dependent upon release of water, stoichiometric considerations, and domain flexibility which all must be taken into account when trying to understand the structural preferences. It may be useful to keep these lessons in mind while attempting to engineer similar structures in the long term. In the short term, a test of our fundamental understanding will be to further probe this system to the degree to be able to determine the minimum number of mutations to generate a rationally designed maxi-ferritin cage from a min-ferritin monomer starting point and vice versa. In conclusion, the hybrid ferritin protein DPS+E, which had previously been shown to be a 12-mer but larger in size than the mini-ferritin Dps, was crystalized and its high resolution crystal structure was obtained. Examination of this crystal structure showed that the tetrahedral symmetry of Dps was retained in the assembled Dps+E cage and that the addition of the Edomain from octahedral Bfr was not sufficient to change the symmetry. Moreover, it was


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determined that the E-domain was forced into a “flipped out” state where it projects away from the nanocage supporting the veracity of the “flip-flop” hypothesis. However, as the electron density at this domain was poorly defined, its entire structure could not be fully determined. It is presumed that the projection of twelve copies of this domain away from the nanocage is the reason the protein was determined to be larger in size than Dps. Through further study of this and other designs in other systems, it is hoped that the fundamentals can be firmly established of why cage proteins with similar tertiary folds assemble into divergent quaternary structures.

ASSOCIATED CONTENT Supporting Information. Supporting information includes cloning and protein purification methods, SDS-PAGE, TEM, SEC and mass spec analysis, as well as a list of primers used, sequencing results, full amino acid sequence of DPS+E, crystallography conditions screened, and RMSD data from the structural alignment. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Contact Information: [email protected]. Present Addresses ∥

School of materials science and engineering, Nanyang Technological University, Singapore.

Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The research at NTU was supported by an SPMS start-up grant, a Singapore Ministry of Education Academic Research Fund Tier 1 Grant (RG 53/06) and B.P.O’s personal salary. At KCL, it was supported by a Marie Curie CIG: PCIG13-GA-2013-618538. T.A.C was sponsored by SINGA and BSE scholarships at NTU and King’s respectively. Y.S. is supported by Chinese Government Scholarship (CGS) and University of the Chinese Academy of Sciences (UCAS). R.J. is supported by a 2013 MOST China-EU Science and Technology Cooperation Program, Grant No. 2013DFE33080, by the National Natural Science Foundation of China (Grant No. 31471238), a 100 talent award of the Chinese Academy of Sciences. Diffraction data were collected at beamline X29 of the National Synchrotron Light Source (NSLS) supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors are grateful to Howard Robinson for data collection and processing. We thank Y. Zhang and M. Ardejani, for insightful conversations. We also thank the crystallographic facility in Biopolis (ASTAR, Singapore) for their help in setting up the crystal screens as well as to the core facilities in NTU for access to the equipment required for characterization.


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ABBREVIATIONS Dps, DNA-binding protein from starved cells; Bfr, Bacterioferritin; SEC, Size exclusion chromatograph; TEM, transmission electron microscopy; DLS, dynamic light scattering; SDSPAGE, sodium dodecyl sulfate poly acrylamide gel electrophoresis. REFERENCES 1. Zhang, Y., Orner, B. P. (2011) Self-assembly in the ferritin nano-cage protein superfamily. Intl. J. Mol. Sci. 12, 5406-5421. 2. Whitesides, G. M., Grzybowski, B. (2002) Self-assembly at all scales. Science 295, 24182421. 3. Dill, K. A., Ozkan, S. B., Shell, M. S., Weikl, T. R. (2008) The protein folding problem. Annul. Rev. of Biophys. 37, 289-316. 4. Ellis, R. J. (1997) Molecular chaperones: Avoiding the crowd. Current Biology 7, R531R533. 5. Kis, K., Volk, R., Bacher, A. (1995) Biosynthesis of riboflavin - studies on the reactionmechanism of 6,7-dimethyl-8-ribityllumazine synthase. Biochemistry 34, 2883-2892. 6. Aisen, P., Listowsky, I. (1980) Iron transport and storage proteins. Annul. Rev. Biochem. 49, 357-393. 7. Harrison, P. M., Arosio, P. (1996) Ferritins: Molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Act. 1275, 161-203. 8. Ardejani, M. S., Li, N. X., Orner, B. P. (2011) Stabilization of a protein nanocage through the plugging of a protein-protein interfacial water pocket. Biochemistry 50, 4029-4037. 9. Ardejani, M. S., Chok, X. L., Foo, C. J., Orner, B. P. (2013) Complete shift of ferritin oligomerization toward nanocage assembly via engineered protein-protein interactions. Chem. Commun. 49, 3528-3530. 10. Llauro, A., Schwarz, B., Kolivatt, R., de Pablo, P. J., Douglas, T. (2016) Tuning viral capsid nanoparticle stability with symmetrical morphogenesis. ACS Nano 10, 8465-8473. 11. Huard, D. J. E., Kane, K. M., Tezcan, F. A. (2013) Re-engineering protein interfaces yeilds copper-inducible ferritin cage assembly. Nature Chemical Biology 9, 169-176. 12. Pulsipher, K. W., Dmochowski, I. J. (2016) Ferritin: Versatile host, nanoreactor, and delivery agent. Isr. J. Chem. 56, 660-670. 13. Fan, R., Chew, S. W., Cheong, V. V., Orner, B. P.(2010) Fabrication of gold nanoparticles inside unmodified horse spleen apoferritin. Small 6, 1483-1487. 14. Douglas, T., Young, M. (1998) Host-guest encapsulation of materials by assembled virus protein cages. Nature 393, 152-155. 15. Abe, S., Maity, B., Ueno, T. (2013) Design of a confined environment using a protein cage and crystals in development of biohybrid materials. Chem Commun. 52 6496-6512. 16. Geninatti-Crich, S., Cadenazzi, M., Lanzardo, S., Conti, L., Ruiu, R., Alberti, D., Cavallo, F., Cutrin, J. C., Aime, S. (2015) Targeting ferritin receptors for the selective delivery of imaging and therapeutic agents to breast cancer cells. Nanoscale 7, 6527-33.


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17. Bhaskar, S., Lim, S. (2017) Engineering protein nanocages as carriers for biomedical applications. NPG Asia Materials 9, e371. 18. Appella, D. H., Christianson, L. A., Karle, I. L., Powell, D. R., Gellman, S. H. (1996) βPeptide foldamers: robust helix formation in a new family of β-amino acid oligomers. J. Am. Chem. Soc 118, 13071-13072. 19. Theil, E. C., (1987) Ferritin - structure, gene-regulation, and cellular function in animals, plants, and microorganisms. Annl. Rev. Biochem. 56, 289-315. 20. Ferreira, C., Bucchini, D., Martin, M.-E., Levi, S., Arosio, P., Grandchamp, B., Beaumont, C. (2000) Early embryonic lethality of H ferritin gene deletion in mice. J. Biol. Chem. 275, 3021-3024. 21. Chiancone, E., Ceci, P., Ilari, A., Ribacchi, F., Stefanini, S. (2004) Iron and proteins for iron storage and detoxification. Biometals 17, 197-202. 22. Theil, E. C., Tosha, T., Behera, R. K. (2016) Solving biology's iron chemistry problem with ferritin protein nanocages, Acc. Chem. Res. 49, 784-791. 23. Fan, R., Boyle, A. L., Cheong, V. V., Ng, S. L., Orner, B. P. (2009) A helix swapping study of two protein cages. Biochemistry 48, 5623-5630. 24. Zhang, Y., Raudah, S., Teo, H., Teo, G. W. S., Fan, R., Sun, X., Orner, B. P. (2010) Alanine-shaving mutagenesis to determine key interfacial residues governing the assembly of a nano-cage maxi-ferritin. J. Biol. Chem. 285, 12078-12086. 25. Cornell, T. A., Fu, J., Newland, S. H., Orner, B. P. (2013) Detection of specific protein– protein interactions in nanocages by engineering bipartite FlAsH binding sites. J. Am. Chem. Soc. 135, 16618-16624. 26. Crichton, R. R., Declercq, J.-P. (2010) X-ray structures of ferritins and related proteins. Biochim. Biophys. Act. 1800, 706-718. 27. Andrews, S. C., Arosio, P., Bottke, W., Briat, J. F., Vondarl, M., Harrison, P. M., Laulhere, J. P., Levi, S., Lobreaux, S., Yewdall, S. J. (1992) Structure, function, and evolution of ferritins. J. Inorg. Biochem. 47, 161-174. 28. Stefanini, S., Chiancone, E., Arosio, P., Finazziagro, A., Antonini, E. (1982) Structural heterogeneity and subunit composition of horse ferritins. Biochemistry 21, 2293-2299. 29. Grant, R. A., Filman, D. J., Finkel, S. E., Kolter, R., Hogle, J. M. (1998) The crystal structure of Dps, a ferritin homolog that binds and protects DNA. Nature Structural Biology 5, 294-303. 30. Chiancone, E., Ceci, P. (2010) The multifaceted capacity of Dps proteins to combat bacterial stress conditions: Detoxification of iron and hydrogen peroxide and DNA binding. Biochim. Biophys. Act. 1800, 798-805. 31. Gerl, M., Jaenicke, R. (1987) Mechanism of the self-assembly of apoferritin from horse spleen. Cross-linking and spectroscopic analysis. Eur. Biophys. J. 15, 103–109. 32. Yariv, J., Kalb, A. J., Sperling, R., Bauminger, E. R., Cohen, S. G., Ofer, S. (1981) The composition and the structure of bacterioferritin of Escherichia coli. Biochem. J. 197, 171-5. 33. Zhang, Y., Fu, J., Chee, S. Y., Ang, E. X. W., Orner, B. P. (2011) Rational disruption of the oligomerization of the mini-ferritin E. coli DPS through protein-protein interface mutation. Protein Science 20, 1907-1917. 34. Cornell, T. A., Orner, B. P. Detection of protein cage assembly with bisarsenic fluorescent probes. In Protein Cages, Orner, B. P., Ed. Springer New York: 2015; Vol. 1252, pp 79-89.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 22

35. Cornell, T. A., Ardejani, M. S., Fu, J., Newland, S. H., Zhang, Y., Orner, B. P. A structure-based assembly screen of protein cage libraries in living cells: Combinatorially repacking a protein-protein interface to recover cage formation in an assembly-frustrated mutant. Manuscript in preparation. 36. Luzzago, A., Cesareni, G. (1989) Isolation of point mutations that affect the folding of the H-chain of human ferritin in Escherichia-coli. Embo J. 8, 569-576. 37. Otwinowski, Z., Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Macromol.r Crystallog. A 276, 307-326. 38. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallog. 40, 658-674. 39. Stein, N. (2008) CHAINSAW: A program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallog. 41, 641-643. 40. Zhang, K. Y., Cowtan, K., Main, P. (1997) Combining constraints for electron-density modification. Meth. Enzymol. 277, 53-64. 41. Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. (2010) Features and development of Coot. Act. Crystallog. D 66, 486-501. 42. Murshudov, G. N., Vagin, A. A., Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Act. Crystallog. D 53, 240-255. 43. Painter, J., Merritt, E. A. (2006) Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Act. Crystallog. D 62, 439-450. 44. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W.; Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., Zwart, P. H. (2010) PHENIX: A comprehensive Python-based system for macromolecular structure solution. Act. Crystallog. D 66, 213-221. 45. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., Ferrin, T. E. (2004) UCSF chimera - A visualization system for exploratory research and analysis. J. Comp. Chem. 25, 1605-1612.


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Mini-Ferritin ... - ACS Publications

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