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.

Tryptophan mutants of intestinal fatty acid-binding protein: ultraviolet absorption and circular dichroism studies.

An UV absorption and CD study of intestinal fatty acid-binding protein is presented. Since there are only two Trp residues in the molecule, two single-Trp mutants were prepared to deconvolute their signals. The individual contribution of the eight Phe and four Tyr residues was not established; however, Phe global contribution is relatively free of interferences from the other chromophores and was observed directly. CD spectra showed that Phe vibronic structure was unusually sharp and seems to monitor very specific details in the three-dimensional structure. The global signal from Tyr was assigned only approximately due to band broadening and overlapping. At the upper end of the CD spectrum, strong positive (1)L(b) Trp transitions from Trp 82 and strong negative (1)L(b) Trp transitions from Trp 6 were observed. (1)L(a) transitions were overall weak, positive for Trp 82 and negative for Trp 6, nearly cancelling each other out in the final spectrum. The above assignment is of practical and fundamental interest to monitor folding, binding, and molecular dynamics down to microdomain resolution. The assignment of Trp bands allowed comparison with previous data from CRABP1, another member of the IFABP family with 28% identical residues. It was found that structural homology extends beyond sequence and tertiary fold to include optical properties of equivalent Trp residues in the structure.