Engineering a compact non-native state of intestinal fatty acid-binding protein.

The last three C-terminal residues (129-131) of intestinal fatty acid-binding protein (IFABP) participate in four main-chain hydrogen bonds and two electrostatic interactions to sequentially distant backbone and side-chain atoms. To assess if these interactions are involved in the final adjustment of the tertiary structure during folding, we engineered an IFABP variant truncated at residue 128. An additional mutation, Trp-6-->Phe, was introduced to simplify the conformational analysis by optical methods. Although the changes were limited to a small region of the protein surface, they resulted in an IFABP with altered secondary and tertiary structure. Truncated IFABP retains some cooperativity, is monomeric, highly compact, and has the molecular dimensions and shape of the native protein. Our results indicated that residues 129-131 are part of a crucial conformational determinant in which several long-range interactions, essential for the acquisition of the native state, are established. This work suggests that carefully controlled truncation can populate equilibrium non-native states under physiological conditions. These non-native states hold a great promise as experimental models for protein folding.