). “Temperature-Induced Conformational Transition of Intestinal Fatty Acid Binding Protein Enhancing Ligand Binding: A Functional, Spectroscopic, and Molecular Modeling Study“

ARIGHI, M.C.; ROSSI, J.P.F.C.; DELFINO, J.M.

BIOCHEMISTRY

American Chemical Society

Lugar: Vanderbilt; Año: 1998 p. 428 - 433

0006-2960

ABSTRACT: Intestinal fatty acid binding protein (IFABP) undergoes a reversible thermal transition between35 and 50 °C, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein,the molar ellipticity measured at 254 nm (possibly implicating the environment around F17 and/or F55)decreases significantly in this temperature range, while in the holoprotein (bound to oleic acid), thisphenomenon is not observed. Concomitantly, an increase in the activity of binding to [14C]oleic acidoccurs. Nevertheless, other spectroscopic evidence indicates that the â-barrel structure, the major motifof this protein, is highly stable up to 70 °C. No changes associated with conformation were detected forboth structures by fourth-derivative analysis of the UV absorption spectra, circular dichroism in the far-UV region, and intrinsic fluorescence measurements. Further structural information arises from experimentsin which binding to the anionic fluorescent probes 1-anilinonaphthalene-8-sulfonic acid (ANS) and itsdimer bisANS was examined. The fluorescence intensity of bound ANS diminishes monotonically, whereasthat of bisANS increases slightly in the temperature range of 35-50 °C. Given the different size of theseprobes, model 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 theseresults, we believe that this subtle conformational transition of IFABP, which positively influences thebinding activity, would involve fluctuations at the peripheral “entry portal” region for the ligand. Thisinterpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidencedby NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 1450-1460].