Computational design of a homotrimeric metalloprotein with a trisbipyridyl core

Abstract

Metal-chelating heteroaryl small molecules have found widespread use as building blocks for coordination-driven, self-assembling nanostructures. The metal-chelating noncanonical amino acid (2,2'-bipyridin-5yl)alanine (Bpy-ala) could, in principle, be used to nucleate specific metalloprotein assemblies if introduced into proteins such that one assembly had much lower free energy than all alternatives. Here we describe the use of the Rosetta computational methodology to design a self-assembling homotrimeric protein with [Fe(Bpy-ala)₃]²⁺ complexes at the interface between monomers. X-ray crystallographic analysis of the homotrimer showed that the design process had near-atomic-level accuracy: The all-atom rmsd between the design model and crystal structure for the residues at the protein interface is ∼1.4 Å. These results demonstrate that computational protein design together with genetically encoded noncanonical amino acids can be used to drive formation of precisely specified metal-mediated protein assemblies that could find use in a wide range of photophysical applications.

Keywords: computational protein design; metalloproteins; noncanonical amino acids; protein self-assembly.

Fig. 1.

An overview of the computational design methods. (A) Octahedral [Fe(Bpy-ala)₃]²⁺ complexes (yellow sticks, Λ and Δ isomers) were generated from small-molecule crystal structures and used as inputs for a docking algorithm that identified sites of incorporation in repeat protein scaffolds (gray cartoons) compatible with threefold symmetric protein complexes. (B) Successfully docked trimers (multicolored cartoons) were compatible with the geometries set by [Fe(Bpy-ala)₃]²⁺ complexes and contained no steric clashes between protein subunits. (C) Computational interface design methods were used to engineer highly complementary surfaces between trimer subunits (light blue sticks) to drive the formation of a protein complex with a desired threefold symmetric orientation.

Fig. S1.

SDS/PAGE analysis of original trimer designs expression. The expression of the designed proteins (TRI_01–TRI_07) and the parent scaffolds (Ank_1–Ank_3) are shown. Above each lane in the designed proteins are a minus sign (indicating that Bpy-ala was not added during expression), a plus sign (indicating that Bpy-ala was added to the expression medium), or a Y (indicating suppression of the amber codon in the designed proteins with a suppressor tRNA charged with tyrosine, which served as an expression control).

Fig. 2.

Experimental characterization of designs TRI_03 and TRI_05. (A, C, and E) Spectroscopic characterization of TRI_03 (A) and TRI_05 (C) and [Fe(Bpy-ala)₃]²⁺ (E) in the range of 230–650 nm is shown. The MLCT regions of these spectra are enlarged in Insets. (B and D) SEC chromatograms of TRI_03 (B) and TRI_05 (D) are shown (solid lines) overlaid with traces of the monomeric parent scaffolds of TRI_03 and TRI_05 (PDB ID codes 4gpm and 4hb5, respectively; dashed lines). Soluble aggregates are observed for each protein at the column void volume of ∼8 mL. (F) Near-UV CD analysis of TRI_05 gave a spectrum consistent with the Λ isomer of a [Fe(Bpy)₃]²⁺ complex.

Fig. 3.

X-ray crystallographic analysis of TRI_05. (A and B) Electron density in the vicinity of the [Fe(Bpy-ala)₃]²⁺ complex of TRI_05 is shown contoured to 1.5 σ. (C) An overlay of the design model (white) with the solved structure (blue) is shown in the vicinity of the designed interface. All designed residues in the interface are depicted as sticks. Residues whose side chains deviated from the designed conformation are labeled.

Fig. 4.

Transient absorbance analysis of TRI_05. (A) Bleaching of TRI_05 MLCT absorbance at seven distinct time delays after excitation at 440 nm. (B) Excited-state lifetime of TRI_05 (red line) and [Fe(Bpy-ala)₃]²⁺ (blue line) complexes after excitation at 440 nm. Absorbance measurements were obtained at 530 nm.

Fig. S2.

Monoexponential fits of transient absorbance data of [Fe(Bpy-ala)₃]²⁺ and TRI_05. Data collected for the free Bpy-ala in complex with Fe²⁺ and TRI_05 (red lines) along with the associated fits (black lines) are shown in A and B, respectively. Fits suggested lifetimes of 610 ps for [Fe(Bpy-ala)₃]²⁺ (A) and 690 ps for TRI_05 (B). These lifetimes are essentially identical within error.

Fig. S3.

CD analysis of TRI_05 stability. (A–C) CD of TRI_05 measured at 25 °C (A), 95 °C (B), and after cooling to 25 °C (C) are shown. (D) The change in CD signal at 220 nm with increasing temperature is shown.

Fig. S4.

Metal removal and concentration-dependent oligomerization of TRI_05. (A) Spectroscopic analysis of [Fe(phen)₃]²⁺ (red line) and [Fe(Bpy-ala)₃]²⁺ (blue line) complexes in the near-UV and visible range (300–600 nm) are shown. (B) Spectroscopic analysis of TRI_05 in the near-UV and visible range is shown before (blue) and after (red) incubation of the protein with Phen at 65 °C. The change in spectral signature in this range suggests the removal of Fe²⁺ from TRI_05 by Phen. (C) SEC analysis of apo TRI_5 (blue line) is shown in comparison to TRI_05 containing Fe²⁺ (red line) and the parent scaffold (black line). The presence of a shoulder on the apo TRI_05 trace suggests partial dissociation of the TRI_05 trimer upon metal removal. (D) Spectroscopic analysis of apo TRI_05. Absorbance is shown in the range of 230–600 nm. An absorbance maximum at 280 nm was observed, which was blue-shifted relative to the spectrum of TRI_05 bound to Fe²⁺. (D, Inset) The MLCT absorbance range is shown. The lack of any signal from 420 to 600 nm suggests that the Fe²⁺ has been removed from the protein complex. (E) Comparison of TRI_05 (red trace) with a mutant in which in which Bpy-ala was replaced with tyrosine (blue trace). Analysis was carried out on a Superdex 75 5/150 analytical column. To further confirm the observation, a comparison with protein standards of known size is shown (black trace). The first peak in the protein standards triplet is BSA with a molecular weight of 66,000 g/mol. (F) The concentration dependence of complex formation was examined. Apo TRI_05 was concentrated to 750 µM and analyzed by SEC (black trace). Fractions collected from this run were then diluted to concentrations of 75 µM (blue trace) and 35 µM (red trace) subjected to gel filtration. The elution volume of the major peak shifts from 14.6 mL in the most concentrated sample to 16.0 mL in the intermediate concentration to 16.6 mL in the least concentrated form, suggesting a concentration dependence of trimer formation. (G) Spectral analysis of apo TRI_05 before (blue line) and after (red line) readdition of Fe²⁺. (H) SEC analysis of apo TRI_05 before (blue trace) and after (red trace) readdition of Fe²⁺.

Fig. S5.

Transient absorbance of [Fe(Bpy-ala)₃]²⁺ complexes collected in air and in an inert environment. Transient absorbance data collected of small-molecule [Fe(Bpy-ala)₃]²⁺ complexes collected in air (red line) and under N₂ (blue line) are shown.