Cooperative deformation of a de novo designed protein

A de novo protein design has been made to understand the uniquepacking of natural proteins that have a ß/-barrelfold. A carefully designed 207 amino acid sequence was synthesizedusing an Escherichia coli expression system and the structuraland thermodynamic characteristics of the purified protein werestudied. At neutral pH the protein is soluble and monomeric,with large amounts of secondary structure and a hydrophobiccore, although the broad resonance peaks of its NMR spectrumsuggest that the designed protein does not have a unique structurewith tightly packed side chains. In an H–D exchange experiment,no amido protons of the designed protein exchanged slowly withdeuterons. At acidic pH, thermal unfolding was observedwitha remarkable change in the excess heat capacity measured directlyby a differential scanning microcalorimeter. The enthalpy andentropy differences at 110°C, extrapolated from analyzedthermodynamic parameters, are 1/3 of the common values for naturalproteins. These measurements indicate that the folding is significantlycooperative as expected, but that the protein is still looselypacked.