Studies on protein structure-function relationship by de novo design

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The de novo designed Cro protein (Isogai, et al., J.Mol.Biol. 354, 801-814, 2005) and its core variants were synthesized to investigate the sequence dependence of the protein folding. The designed Cro proteins unfold in a non-cooperative, anomalous manner with temperature increased, whereas the native lambda phage Cro protein exhibits cooperative unfolding, in which the secondary structures collapse in the identical time courses. In the designed proteins, α helices gradually disrupted slower than β sheets. The degree of the folding non-cooperativety, or stability of folding intermediate, correlates with the tendency to form insoluble protein aggregate or amyloid fibrils. The present results indicate that amino acid sequences exhibiting cooperative folding can be further selected from the sequences that fold into a well-defined structure, and that the native-like folding properties represent one of the driving forces in naturally occurring sequence selection. Native proteins may have acquired their folding cooperativity under selective pressure to avoid formation of the toxic aggregate or amyloid fibrils in the molecular evolution.