To better understand how amino acid sequence encodes protein structure, we engineered mutational pathways that connect three common folds (3α, β−grasp, and α/β−plait). The structures of proteins at high sequence-identity intersections in the pathways (nodes) were determined using NMR spectroscopy and analyzed for stability and function. To generate nodes, the amino acid sequence encoding a smaller fold is embedded in the structure of an ~50% larger fold and a new sequence compatible with two sets of native interactions is designed. This generates protein pairs with a 3α or β−grasp fold in the smaller form but an α/β−plait fold in the larger form. Further, embedding smaller antagonistic folds creates critical states in the larger folds such that single amino acid substitutions can switch both their fold and function. The results help explain the underlying ambiguity in the protein folding code and show that new protein structures can evolve via abrupt fold switching.

为了更好地理解氨基酸序列如何编码蛋白质结构，我们设计了连接三个常见折叠（3α、β-grasp 和 α/β-plait）的突变途径。使用核磁共振波谱确定通路（节点）中高序列同一性交叉处的蛋白质结构，并分析其稳定性和功能。为了生成节点，编码较小折叠的氨基酸序列被嵌入约 50% 较大折叠的结构中，并设计了与两组天然相互作用兼容的新序列。这会生成较小形式具有 3α 或 β-grasp 折叠但较大形式具有 α/β-plait 折叠的蛋白质对。此外，嵌入较小的拮抗折叠在较大的折叠中产生临界状态，使得单个氨基酸取代可以改变它们的折叠和功能。这些结果有助于解释蛋白质折叠代码中潜在的模糊性，并表明新的蛋白质结构可以通过突然的折叠转换而进化。