H-1, C-13 AND N-15 ASSIGNMENTS AND CHEMICAL SHIFT-DERIVED SECONDARY STRUCTURE OF INTESTINAL FATTY-ACID-BINDING PROTEIN

Sequence-specific H-1, C-13 and N-15 resonance assignments have been established for rat intestinal fatty acid-binding protein complexed with palmitate (15.4 kDa) at pH 7.2 and 37 degrees C. The resonance assignment strategy involved the concerted use of seven 3D triple-resonance experiments (CC-TOCSY, HCCH-TOCSY, HNCO, HNCA, N-15-TOCSY-HMQC, HCACO and HCA(CO)N). A central feature of this strategy was the concurrent assignment of both backbone and side-chain aliphatic atoms, which was critical for overcoming ambiguities in the assignment process. The CC-TOCSY experiment provided the unambiguous links between the side-chain spin systems observed in HCCH-TOCSY and the backbone correlations observed in the other experiments. Assignments were established for 124 of the 131 residues, although 6 of the 124 had missing amide H-1 resonances, presumably due to rapid-exchange with solvent under these experimental conditions. The assignment database was used to determine the solution secondary structure of the complex, based on chemical shift indices for the H-1(alpha), C-13(alpha), C-13(beta), and (CO)-C-13 atoms. Overall, the secondary structure agreed well with that determined by X-ray crystallography [Sacchettini et al. (1989) J. Mol. Biol., 208, 327-339], although minor differences were observed at the edges of secondary structure elements.