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Apo-IFABP and Oleate: A Protein-Small Organic Molecule Interaction

Intestinal fatty acid-binding protein (IFABP) belongs to a family of @15 kDa, predominately b-sheet proteins that bind a diverse group of polar lipids [49]. IFABP

consists of a helix-turn-helix motif and a b-clam topology surrounding a large cavity into which a single fatty acid binds, permitting intracellular trafficking and processing of dietary fatty acid in the intestine [50, 51]. A D34A-IFABP mutant with a single-site mutation by replacing Asp 34 with Ala is available, and the structure of its apo-form was solved by Ogbay and Cistola using NMR [52]. H/D exchange kinetics and PLIMSTEX applied to wild-type rat IFABP (WT-IFABP) and its D34A mutant (D34A-IFABP) add information on the differences in conformation and fatty acid-binding properties of this protein [53, 54].

The PLIMSTEX curve for 0.3 mM WT-IFABP titrated with potassium oleate fits well with a 1:1 binding model [22, 24]. The K and DD1 (difference between the average deuterium level of one-ligand-bound protein and that of apo protein) for WT-IFABP are (2.6 + 0.6) x 106 M—1 and 13.8 + 0.7 (Table 11.1), respectively, indicating that a strong interaction between oleate and WT-IFABP occurs with protection of @14 backbone amide hydrogens.

The titration curve for 3 mM D34A-IFABP with oleate is more complicated and fits well a 1:3 protein:ligand sequential binding model [54]. The binding affinities of each oleate to D34A [Kd; (i = [email protected]) are between 6 mM and 140 mM] are lower than that of the single binding constant for WT-IFABP [0.38 mM]. Complexes with one and three oleates are more hydrogen-bonded than those of apo D34A-IFABP (DD1 = 25; DD3 = 32); however, the two-oleate bound D34A protein is less hydrogen-bonded than apo (DD2 = —9). Mass profiles obtained in an FTMS experiment showed that multiple components form during oleate titration. For example, the pattern of peaks representing uptake of deuterium by D34A-IFABP when the ratio of oleate to D34A is 15:1 fits three binomial distributions.

These results demonstrate that the disruption of the D34-R126 ion pair in IFABP causes the D34A mutant to bind additional oleates (at least three) with lower binding constants than for WT-IFABP binding the first oleate.

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