A DESIGNED 4-HELIX BUNDLE PROTEIN WITH NATIVE-LIKE STRUCTURE

A 108 amino acid protein was designed and constructed from a reduced a lphabet of seven amino acids. The 2.9 Angstrom resolution X-ray crysta l structure confirms that the protein is a four helix bundle, as it wa s designed to be. Hydrogen/deuterium exchange experiments reveal burie d amide protons with protection factors in excess of 1 x 10(6) in the range characteristic of well protected protons in functional folded pr oteins (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 excha nge 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 de grees C, consistent with the free energy of folding derived from the p rotection factors of the most protected protons, indicating that globa l unfolding is required for exchange of the most protected protons.