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. 2016 Jan;12(1):29-34.
doi: 10.1038/nchembio.1966. Epub 2015 Nov 23.

De Novo Design of a Four-Fold Symmetric TIM-barrel Protein With Atomic-Level Accuracy

Free PMC article

De Novo Design of a Four-Fold Symmetric TIM-barrel Protein With Atomic-Level Accuracy

Po-Ssu Huang et al. Nat Chem Biol. .
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Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of four-fold symmetrical (β/α)8 barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of side chain-backbone hydrogen bonds for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical to that of the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has sampled only a subset of the sequence space available to the TIM-barrel fold. The ability to design TIM barrels de novo opens new possibilities for custom-made enzymes.


Figure 1
Figure 1. Geometric constraints on the secondary structure arrangement in an ideal 4-fold symmetric TIM-barrel
(a) The asymmetric β-strand arrangement of the classic triosephosphate isomerase barrel from chicken muscle (PDB code: 1TIM). The strands, as defined by DSSP, are viewed from the inside of the barrel, with open and shaded circles representing amino acid residues pointing into and out of the barrel, respectively. The first strand is shown at the left and right. Three horizontal lines indicate the residues lining the interior of the barrel. Sheet hydrogen bonds follow diagonal directions indicated by dashed red lines. (b, c, d) The three solutions for achieving the s = 8 shear with 4-fold symmetry. Orange boxes represent 4 individual repeat units; the first unit is highlighted for clarity. Since residues represented by open and shaded circles are structurally non-equivalent and the diagonal running hydrogen bonds must connect residues pointing in the same direction, an 8-fold symmetric barrel is not possible. (b) Register shift of one residue between every strand. (c) Register shift alternating between 0 and 2 residues around the barrel, with strands starting with residues pointing towards the helices (shaded circles). (d) Alternating (0,2) register shift as in c., with strands starting with residues pointing into the barrel (open circles). (e) To achieve the shear pattern in d, the helix spanning the offset strands (yellow) must be shorter than the helix within the repeat unit.
Figure 2
Figure 2. Sequence determinants of de novo designed TIM-barrel
Designed TIM-barrel model is depicted with light green circles tagging regions shown in the insets, where the design models are shown in pink and X-ray structures in blue. Residues are numbered by the design model, but X-ray structure residue numbers are in parentheses. (a) The α/β loop at the interface between the repeat units with a register shift of 2. Asp1 was designed to satisfy the hydrogen bonding requirement for the backbone amide group on the neighboring strand. In the crystal, Arg21 makes lattice contacts rather than the designed interaction; in solution the designed hydrogen bond may be formed. (b) Features stabilizing the β/α loop backbone. Ser32 was designed to interact with the amide group that points towards the hydrophobic core, and Gln62 with the carbonyl similar to Arg21 (in a). Alternative conformations of Gln62 are observed in two different repeats in the crystal structure. (c) Packing of the sheet facing side of the helices (long helix, white; short helix, yellow) against the surface on the sheet. (d) The wedges between the helices are filled by tryptophans. Trp42 was found to adopt a different conformation in the crystal structure. (e) Trp42 was designed to interact with Thr26 on the neighboring loop directly, but crystallographic evidence suggests that the same interaction is mediated by water, as shown by the clear electron density bridging the two residues.
Figure 3
Figure 3. Effect of two vs. four-fold symmetrical barrel interior on stability
Starting from the sTIM-1 sequence, which carried most of the stabilizing features, we explored variations in the interior of the β-barrel. The different packing layers of the barrel are shown with circles in the central barrel figure. Models for two of the layers where asymmetric designs were made are shown in red and orange. Two configurations were tested for the polar red layer, and three for the hydrophobic orange layer. The six possible combinations were tested with variants sTIM-1~6. For example, sTIM-1 is 4-fold symmetrical with four arginine-aspartate salt-bridges crowning the top (red layer) and has four isoleucines in the interior of the barrel (orange layer). Strands are sequentially colored from blue to red, and for the orange layer configurations, sidechain packing are shown with space-fill spheres. Stabilities of the six different variants correlate strongly with the configurations in the hydrophobic packing layer. sTIM-1 (71.9 °C) ≈ sTIM-4 (71.8 °C) > sTIM-2 (66.5 °C) ≈ sTIM-5 (65.8 °C) > sTIM-6 (59.3 °C) ≈ sTIM-3 (56.9 °C). (CD melting curves of these variants are in Supplementary Fig. 5)
Figure 4
Figure 4. Stability and structure of sTIM-11
(a) Superposition of the X-ray crystal structure (in blue) and the design model (in pink). (b) Comparison of the crystal structure and design model over two internal repeat units (residues 47-92 and 93-138). The internal repeats are nearly identical, and their side chains are in perfect agreement with the model. (c) Chemical denaturation with guanidinium chloride (GdmCl) followed by CD (open circles) and fluorescence (closed circles). Signals for the secondary (CD) and tertiary (fluorescence) structures are lost simultaneously at ~2 M GdmCl. (d) Thermal melt followed by CD. (e) CD wavelength spectra of sTIM-11 at 30 °C (line), when melted at 95 °C (dashed), and after cooling at 30°C (dotted).
Figure 5
Figure 5. Sequence relationships between natural (β/α)8-barrels and sTIM-11
HHsearch calculations with sTIM-11 against the Astral SCOPe 2.04 database filtered for 95% sequence identity were carried out, and clustering was performed at a P-value cutoff of 1.0e–02. Connections are shown in different shades of grey with a linear scaling between P-values of 1.0e–02 and 1.0e–56.

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