Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1')anilinonaphthalene binding to intestinal fatty acid binding protein

Abstract

1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity.

Characterization of the Binding of Drugs to Human Intestinal Fatty Acid Binding Protein (IFABP): Potential Role of IFABP as an Alternative to Albumin for in Vitro-in Vivo Extrapolation of Drug Kinetic Parameters

“…The binding of ANS was well described by the single binding site equation. The derived dissociation constant (2.9 M) is comparable with K d values reported previously for the unmodified rat protein (6.9 M, Kirk et al, 1996;3.6 -7.4 M, Velkov et al, 2005). Scatchard analysis indicated that each molecule of IFABP bound one molecule of ANS.…”

Conformational Changes and Stabilization Induced by Ligand Binding in the DNA-binding Domain of the E2 Protein from Human Papillomavirus

“…In principle, it is possible that it binds to the DNA binding site and at the central cavity of the barrel, or some other accessible non-polar site. The presence of more than one site is supported by the biphasic changes in the wavelength maximum, indicative of sites of different polarity (28). To complement our conformational analysis, we used the extreme sensitivity of bis-ANS to investigate conformational changes caused by ionic strength, phosphate, DNA, and heparin on the E2 domain.…”

The protective effect of crocin on the amyloid fibril formation of aβ42 peptide in vitro

“…ANS is a hydrophobic fluorescent molecular probe that has been used for examining the non-polar character of proteins and membranes [23][24][25]. ANS binding is used to study changes in exposed hydrophobicity in Aβ 42 upon incubation in the presence and absence of crocin.…”