Structure of a designed protein cage that self-assembles into a highly porous cube - PubMed (original) (raw)

Structure of a designed protein cage that self-assembles into a highly porous cube

Yen-Ting Lai et al. Nat Chem. 2014 Dec.

Abstract

Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here, we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Å diameter, cube-shaped cage with large openings into a 130 Å diameter inner cavity. A crystal structure of the cage showed atomic-level agreement with the designed model, while electron microscopy, native mass spectrometry and small angle X-ray scattering revealed alternative assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will probably require limiting flexibility to select against alternative forms. These results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.

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Figures

Figure 1

Figure 1. Models of the engineered fusion protein and its assembled cage structure

a, The designed fusion protein, with trimeric KDPGal aldolase (green), the 4-residue helical linker (blue), and the dimeric domain of FkpA protein (orange) shown with lines for the 3-fold (cyan) and 2-fold (magenta) symmetry axes. b, A model of the intended 24 subunit cage with octahedral symmetry in a bounding box (left). The three-fold symmetry axes (cyan) and two-fold symmetry axes (magenta) of a cube are shown on the right.

Figure 2

Figure 2. Crystal structure of the designed cubic cage ATC-HL3

a, Four complete ATC-HL3 cages, colored differently, are packed within a unit cell (shown in blue lines). b, The cube-shaped cage observed in the crystal matches the design with high accuracy. The crystal structure is shown in green ribbon and the intended design is shown in cyan ribbon; the two are nearly perfectly overlapping. c, The packing alignment of cages in the crystal produces a highly porous protein lattice; a 3×3 block of unit cells is shown. The two independent protein chains in the asymmetric unit are colored differently (purple or green).

Figure 3

Figure 3. Native mass spectrometry of the ATC-HL3 protein cage

a, The native mass spectrum (top) indicated three major assembly forms composed from 12, 18, and 24 subunits. The sizes of these three major assembly species were confirmed by a tandem mass spectrometry analysis (below) in which the masses were examined after stripping one subunit from each assembly form to give (n-1) subunits. A trimeric species was also evident in the native mass spectrum (top). b, Hypothetical models are shown of the three major assembly forms observed: atetrahedron (top), a triangular prism (middle), and the cube (bottom). The cube form shown is the intended computation design, while the two smaller forms were constructed by rigid body positioning of dimers and trimers allowed by minor bending at the linkers between protein domains. Trimers occupying different vertices are color-coded differently.

Figure 4

Figure 4. Negatively stained transmission electron microscopy of the ATC-HL3 cage

a, A wide field view showing assembled protein particles preserved in stain. b, Examples of 2D class averages (left) obtained after aligning, clustering, and averaging similar particle images. For comparison to what would be expected for EM images of the designed protein assembly, calculated projections (middle) are shown of models of the 24-subunit cube assembly (the intended design) and an alternate 12-subunit tetrahedral assembly (both calculated using a 4 nm low-pass image filter). Three dimensional atomic models are also shown (right).

Figure 5

Figure 5. SAXS profile of ATC-HL3

a, The observed small angle X-ray scattering (SAXS) profile is shown (black) for the designed protein (in 600 mM ammonium sulfate), along with a weighted sum of SAXS profiles calculated from atomic models of the designed (24-mer) cube-shaped assembly and the 18-mer and 12-mer assemblies, all in their flexed forms. The Guinier region is shown in the inset; its near-linearity indicating the absence of other aggregated forms of the protein. A residual error plot (bottom), expressed as a ratio of the observed:calculated scattering intensity (with ideal value of 1) is shown at the bottom. The close agreement between the calculated and observed profiles supports the modeling of the assembly in solution as a mixture of the 12-mer, 18-mer, and 24-mer assembly forms. b, Microcrystal peaks observed on the SAXS profile after temperature annealing of a low salt (50 mM ammonium sulfate) condition. The scattering angle of the dominant peak (q=0.044 Å-1) corresponds to the first diffraction peak in the crystal form obtained for the 24-subunit cube (Supplementary Material).

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References

    1. Whitesides GM, Grzybowski B. Self-assembly at all scales. Science. 2002;295:2418–2421. - PubMed
    1. Goodsell DS, Olson AJ. Structural symmetry and protein function. Annu Rev Biophys Biomol Struct. 2000;29:105–153. - PubMed
    1. Lucon J, Qazi S, Uchida M, Bedwell GJ, La France B, Prevelige PE, et al. Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading. Nat Chem. 2012;4:781–788. - PMC - PubMed
    1. Douglas T, Young M. Viruses: Making friends with old foes. Science. 2006;312:873–875. - PubMed
    1. Huard DJ, Kane KM, Tezcan FA. Re-engineering protein interfaces yields copper-inducible ferritin cage assembly. Nat Chem Biol. 2013;9:169–176. - PubMed

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