A designed four helix bundle protein with native-like structure.

A 108 amino acid protein was designed and constructed from a reduced alphabet of seven amino acids. The 2.9 A resolution X-ray crystal structure confirms that the protein is a four helix bundle, as it was designed to be. Hydrogen/deuterium exchange experiments reveal buried amide protons with protection factors in excess of 1 x 10(6) in the range characteristic of well protected protons in functional folded proteins (10(3)-10(8)) rather than protons in rapid exchange (0-10(2)). The protein is monomeric at 1 mM, the concentration at which the exchange experiments were undertaken, indicating that the exchange factors are due to a unique stable tertiary structure fold, and not due to any higher order quaternary structure. Thermodynamic analysis provides an estimate of the free energy of folding of -9.3 kcal mole-1 at 25 degrees C, consistent with the free energy of folding derived from the protection factors of the most protected protons, indicating that global unfolding is required for exchange of the most protected protons.