De novo design of metal-oxide templating proteins

Abstract

Protein design now enables the precise arrangement of atoms on the nanometer length scales of inorganic crystal nuclei, opening up the possibility of templating the growth of metal oxides including semiconductors. We designed proteins presenting regularly repeating interfaces containing functional groups that organize ions and water molecules, and characterized their ability to bind to and template metal oxides. Two interfaces promoted the growth of hematite under conditions that otherwise resulted in the formation of magnetite. Three interfaces promoted ZnO nucleation under conditions where traditional ZnO-binding peptides and control proteins were ineffective. Designed cyclic assemblies with these ZnO nucleating interfaces lining interior cavities promoted ZnO growth within the cavity. CryoEM analysis of a designed octahedral nanocage revealed atomic density likely corresponding to the growing ZnO directly adjacent to the designed nucleation promoting interfaces. These findings demonstrate that designed proteins can direct the formation of metal oxides not observed in biological systems, opening the door to protein-semiconductor hybrid materials.

Fig. 1:. Design principles for templating mineral growth.

(A) Three hypothesized mechanisms of mineral binding: charge complementarity, metal coordination, and water ordering. (B-D) 36 protein backbones used in the library. Orange spheres show alpha carbons of residues in a designed interface. Scaffolds include 11 designed helical repeats (DHR) (B), 6 alpha-beta topologies (C), and 17 designed beta solenoids (DBS) and 2 native solenoids (in green) (D). (E-N) Example designed surfaces defined by 10 (out of 23) amino acid compositions applied to the DHR backbone DHR14. The colors of the amino acid side chains correspond to chemical categories identified in rectangles. Compositions include highly charged residues (E-H), charged and hydrophobic moieties (I, J), metal chelating residues (K, L), and non-charged hydrophobic residues (M, N). (O-Q) Illustration of the designs generated for each combination of scaffold and composition. (O) Two sets of interface residues are selected on opposing surfaces of protein. For a given surface and amino acid composition, two repeat surfaces (P), with the same residue in analogous positions in each repeat subunit, and two non-repeat surfaces (Q), wherein residues are scrambled among repeats. (R) 4 designed interfaces for each of 2 surfaces yields 8 sequences per combination of 36 protein backbones and 23 amino acid composition, resulting in a library of approximately 7 × 10³ designs.

Fig. 2.. Mineral binding screen.

(A) Illustration of screening strategy. (B) SSC-A vs. FSC-A signals of yeast populations before and after ZnO enrichment sorting. Plots are kernel density plots estimates from 10,000 events. (C) SSC-A signals of a sample sublibrary before and after sorting, and yeast clones displaying three enriched designs vs. control yeast. (D-E) Z0-fiber and Z3 interfaces and mutational analysis of Zn⁺² ion binding with ratiometric dye (Mag-Fura-2). Although mutants are named by the first residue that is mutated, all repeated instances of these residues, circumscribed by lines on the models, are mutated as well. (F) Model of the Z4 enriched design with circles highlighting threonines repeated in pattern resembling TXXXAXXXAXX motif in native antifreeze proteins (AFP). (G) Isothermal titration calorimetry analysis of Zn⁺² against three designed proteins, lysozyme, and solutions without protein (protein concentration = 30 μM). (H) Example native ice-binding proteins: beta solenoid Tenebrio molitor AFP (TmAFP) containing threonine rich parallel beta-sheets (PDBid: 1ezg) and alpha-helical winter flounder AFP (WfAFP) with threonines in TXXXAXXXAXX motif circled (PDBid: 1wfa). (I) H7 and H9 interfaces and analysis of Co⁺² ion binding with ratiometric dye (Mag-Fura-2). Colors of all amino-acid side chains correspond to the chemical categories defined in Figure 1.

Fig. 3.. Characterization of metal oxides nucleation by designed interfaces.

(A) Schematic illustrating the nucleation assay and motif grafting methodology. (B) Emission spectra (Ex. = 325 nm) of solutions superstatured for ZnO seeded with selected ZnO-binding designs compared to ZnO NP, lysozyme and neat (no-additive) solution controls. (C) X-Ray diffraction analysis of synthesized products following two hour incubation with designed proteins (Z0-fiber, Z3, Z4), control protein (lysozyme), or solutions without additive (neat). A reference spectra taken of ZnO nanoparticles (ZnO NP) is also provided. (D) Plot of PL emission over time (Ex. 325, Em. 600) of supersaturated solutions containing selected designs, lysozyme, ZnO NP, or neat solutions. (E) Models of interface motifs taken from Z0 and four Z0-grafted alpha-beta (zab) designs. (F) Models of the Z4 interface motifs with threonine side chains resembling TXXXAXXXAXX motifs observed in native α-helical ice-binding proteins circled in one repeat. (G) Designed Ice-binding Twistless Helical Repeat (iTHR) with threonines in TXXXAXXXAXX motifs circled in one repeat. Colors of amino acid side chains in all panels correspond to the chemical categories defined in Figure 1. (H-I) PL emission over time (Ex. 325, Em. 600) of the zab redesigns vs. Z0-fiber and controls (H) and b-iTHR-201 vs. Z4 and controls (I). (J-K) Box plots showing 6 replicates of growth experiments using designed proteins, controls, and four previously reported ZnO-binding peptides, referred to by the last names of the first authors of the source publications (Umetsu, Tomczak, Wei, and Golec)^–. (J) Average maximum PL intensity (n=6). (K) Nucleation times, defined as the halfway point between the initial and maximum PL signal, for the subset of samples that showed a PL signal of nucleation (maximum PL > 100), n values as indicated. All ZnO growth solutions contain 3 mM ZnNO₃, 50 mM NaCl, 100 mM HEPES pH 8.2, and contain 0.1 mg/mL protein or 13 μg/mL ZnO NP as indicated. (L) Digital scans taken of iron oxide growth reactions over time with protein (0.15 mg /mL) or NP (1.6×10⁻⁵ g/ml Hematite NP; 1.5×10⁻⁵ g/ml Magnetite NP) additives as indicated. Solutions contain ammonium iron(II) sulfate hexahydrate 0.05 mM, Iron(III) nitrate nonahydrate 0.1 mM, KNO₃ 50 mM, CHES 100 mM. Normalized red-intensity of the reactions shown in panel L. Values shown are the mean red intensity divided by the sum of the mean read, mean green, and mean blue intensity within the area of each well (n=6). (N) Raman spectroscopy of the products of the H7-neg, H9-neg, lysozyme, and neat reactions shown in panels L and N, with spectra taken of reference hematite and magnetite nanoparticles for comparison. (O) Images from iron substrate scans prior and following two hours incubation with 10ul samples containing H7-neg (0.6 mg/ml), Lysozyme (0.6 mg/ml) or buffer only.

Fig. 4.. ZnO nucleation by Z4 motif containing designed oligomers.

(A-D) Cyclic oligomers with Z4 motifs lining interior cavities. (A-B) Two C3 designs with design models colored by subunit with Z4 motifs shown as sticks. Design model backbones shown in white superimposed with X-ray crystal structures in blue (PDB IDs: 9d92, 9cc4). Images at bottom right are representative ns-TEM picked particles (left) and 2D class averages (right). (C) Z4-C3i-XL model superimposed with ns-TEM 3D reconstruction, and examples of picked particles and 2D classes. (D) Z4-C6 colored by subunit with motifs shown as sticks, model superimposed with ns-TEM 3D reconstruction, and examples of picked particles and 2D classes. (E) Model of the mutated Z4 knock out (Z4-KO) interface with threonines replaced with glutamates. (F) Plot of PL emission over time (Ex. 325, Em. 600) of Z4-motif containing cyclic oligomers compared to oligomers with interface KO mutants and the Z4 monomer (*sample replicated from Figure 3). (G) Octahedral cage designed using Z4-C3i, ns-TEM picked particles and 2D class averages, and model superimposed with ns-TEM 3D reconstruction. (H) Z4-tube colored by subunit with Z4 motifs shown as sticks, ns-TEM picked particles, 2D-averages, and 3D reconstructions superimposed with models. (I-J) Effect of Z4-motif containing oligomers on ZnO nucleation compared to Z4 monomer, ZnO NP, and neat solutions (*samples are replicated from Figure 3). (I) Maximum PL intensity after nucleation assay (n=6). (J) Nucleation times, defined as the halfway point between the initial and maximum PL signal (n=6), for samples that showed a signal of nucleation (maximum PL > 100). Colors of amino acid side chains correspond to the chemical categories defined in Figure 1. All scale bars are 4 nm.

Fig. 5.. Cryo-EM characterization of ZnO mineralized Z4-cage.

(A) Representative denoised micrograph of Z4-cage particles imaged in the presence of 3 mM Zn(NO₃)₂ for ~30 minutes prior to freezing. Highlighted are examples of “empty” (teal), “filled” (maroon), “blebbed” (purple), and “bridged” (orange) nanoparticle morphologies observed in a single micrograph. (B) Additional examples of each morphology observed in the dataset. (C) 2D class averages of Z4-cage. (D) 3.8 Å cryo-EM 3D reconstruction of Z4-cage refined with relaxed octahedral symmetry. (E) Comparison of the final built cryo-EM model of Z4-cage (grey) with the computational design model (blue). (F) C1 reconstruction of Z4-cage, low-pass filtered to 8 Å, showing ZnO nucleation localized along the C3 symmetry axis (blue), and no ZnO density along the C4 axis (red). (G) Octahedral symmetry expansion and 3D classification of all Z4-C3i trimers from all Z4-cage nanoparticles show both nucleated (blue) and empty (red) trimers exist within the data. (H) Near-atomic resolution 3D reconstruction of nucleated Z4-C3i trimers in the Z4-cage nanoparticle, highlighting density from coordinated H₂O or ions (blue) near the designed ice-binding motif. (I) Low-pass filtered view of (H), zoomed in on the C3 axis, illustrating stochastic ZnO growth within the Z4-C3i trimer. (J) Placement of atoms into the density (see Methods) suggests that initial H₂O/Zn ion coordination and nucleation occurs along the designed Z4 ice-binding motif (sticks).