Temperature-induced conformational transition of intestinal fatty acid binding protein enhancing ligand binding: A functional, spectroscopic, and molecular modeling study

Intestinal fatty acid binding protein (IFABP) undergoes a reversible therma l transition between 35 and 50 degrees C, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein, the molar elliptic ity measured at 254 nm (possibly implicating the environment around F17 and /or F55) decreases significantly in this temperature range, while in the ho loprotein (bound to oleic acid), this phenomenon is not observed. Concomita ntly, an increase in the activity of binding to [C-14]oleic acid occurs. Ne vertheless, other spectroscopic evidence indicates that the beta-barrel str ucture, the major motif of this protein, is highly stable up to 70 degrees C. No changes associated with conformation were detected for both structure s by fourth-derivative analysis of the UV absorption spectra, circular dich roism in the far-UV region, and intrinsic fluorescence measurements. Furthe r structural information arises from experiments in which binding to the an ionic fluorescent probes 1-anilinonaphthene-8-sulfonic acid (ANS) and its d imer bisANS was examined. The fluorescence intensity of bound ANS diminishe s monotonically, whereas that of bisANS increases slightly in the temperatu re range of 35 -50 degrees C. Given the different size of these probes, mod el building suggests that ANS would be able to sense regions located deeply inside the cavity, while bisANS could also reach the vicinity of the small helical domain of this protein. In light of these results, we believe that this subtle conformational transition of IFABP, which positively influence s the binding activity, would involve fluctuations at the peripheral "entry portal" region for the ligand. This interpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidenced by NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 14 50-1460].