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)