Fig. 1 Strategy to capture a transition state. (A) The orientations and relative changes in energy (ΔGrel) between the ground (Φ = ~45°) and transition states (Φ = 0° and 90°) of rotation around the central bond of biphenyl are shown. (B) An overview of the computational design process used to generate candidate designs is shown.
Fig. 2 X-ray crystallographic analysis of BIF_1. (A) The x-ray crystal structure of BIF_1 is shown in yellow; BiPhe and surrounding residues are shown in sticks. Rings A and B are those closest to and farthest from the protein backbone, respectively. Electron density around the BiPhe side chain is shown as a 2Fo – Fc map contoured to 2σ. (B) A comparison of the design model (gray) to the structure (yellow) of BIF_1 is shown; BiPhe, Trp42, and Trp81 are shown in sticks. A loop corresponding to residues 81 to 89 of the parent scaffold is shown in red. Missing density in the structure corresponding to residues 83 to 86 of BIF_1 is shown as a dashed red line. (C) A comparison of the structure of BIF_1 (yellow) to the design model (gray) is shown. BiPhe, Trp42, and Trp81 are shown in sticks. An arrow indicates rotation about χ2 in the structure relative to the design.
Fig. 3 A comparison of the crystal structures of BIF_1.1 to BIF_1.4. (A to D) Crystal structures of second-round mutants BIF_1.1 to BIF_1.4 are shown. The side chains of BiPhe, and those at positions 79 and 123, are shown in sticks. Electron density from a 2Fo – Fc map contoured to 1.5σ [(A to C)] and 2.0σ (D) is shown for the aforementioned residues. The measured dihedral angle between the two biphenyl rings is shown beneath the biphenyl side chain in each case.
Fig. 4 X-ray crystal structure of BIF_0. (A) The crystal structure of BIF_0 is shown in blue, and BIF_1.3 is shown in gray. The BiPhe side chain and surrounding residues are shown in sticks. (B) Packing interactions between the designed protein BIF_0 and the BiPhe side are highlighted with space-filling representations of the interacting residues. (C and D) The structure of BIF_0 is shown, highlighting the BiPhe side chain; views from the front and side are shown. The BiPhe side chain and surrounding residues are shown in sticks, and 2Fo – Fc maps are contoured to 2σ in each case.
- Table 1 First-round computational designs.
Designed protein names, parent scaffolds, and mutations made are listed. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
Designed
proteinParent scaffold
(PDB ID)Computationally designed
mutationsBIF_1 1y2q S8A, I11BiPhe, F42W, Y79A, F81W, K121I, F123Y BIF_2 1anu V20G, F22I, C33W, F35BiPhe, F37W, F96G BIF_3 2h2h F48L, F60BiPhe, Y61W, F63W, A64M, I68G, P157G, I159A BIF_4 1ve0 I21T, V25BiPhe, S26A, V39G, C46A, I48V - Table 2 Second- and third-round crystallographic analysis.
Second-round mutant identities, biphenylalanine dihedral angle, and x-ray crystal structure resolution are listed. Dihedrals listed are averages of those measured on each side of the biphenyl ring.
Scaffold F42 Y79 F123 BIF Φ (deg.) Resolution (Å) BIF_1 W A Y 26 1.95 BIF_1.1 F S V 35 2.10 BIF_1.2 F V V 21 2.50 BIF_1.3 F S A 15 2.36 BIF_1.4 F V A 20 2.10 BIF_0 F I A 0 2.05
Supplementary Materials
www.sciencemag.org/content/347/6224/863/suppl/DC1
Materials and Methods
Figs. S1 and S2
Tables S1 and S2
References (34–42)
Additional Files
- Trapping a transition state in a computationally designed protein bottle
Aaron D. Pearson, Jeremy H. Mills, Yifan Song, Fariborz Nasertorabi, Gye Won Han, David Baker, Raymond C. Stevens, Peter G. Schultz
Materials/Methods, Supplementary Text, Tables, Figures, and/or References
- Materials and Methods
- Figs. S1 and S2
- Tables S1 and S2
- References
