Thermodynamic analysis of ANS binding to partially unfolded α‐lactalbumin: correlation of endothermic to exothermic changeover with formation of authentic molten globules

Abstract

A fluorescent reporter, 8-anilino-1-naphthalene sulfonic acid (ANS), can serve as a reference molecule for conformational transition of a protein because its aromatic carbons have strong affinity with hydrophobic cores of partially unfolded molten globules. Using a typical calcium-binding protein, bovine α-lactalbumin (BLA), as a model protein, we compared the ANS binding thermodynamics to the decalcified (10 mM EDTA treated) apo-BLA at two representative temperatures: 20 and 40 °C. This is because the authentic molten globule is known to form more heavily at an elevated temperature such as 40 °C. Isothermal titration calorimetry experiments revealed that the BLA-ANS interactions at both temperatures were entropy-driven, and the dissociation constants were similar on the order of 10(-4) M, but there was a dramatic changeover in the binding thermodynamics from endothermic at 20 °C to exothermic at 40 °C. We believe that the higher subpopulation of authentic molten globules at 40 °C than 20 °C would be responsible for the results, which also indicate that weak binding is sufficient to alter the ANS binding mechanisms. We expect that the thermodynamic properties obtained from this study would serve as a useful reference for investigating the binding of other hydrophobic ligands such as oleic acid to apo-BLA, because oleic acid is known to have tumor-selective cytotoxicity when complexed with partially unfolded α-lactalbumin. Copyright © 2016 John Wiley & Sons, Ltd.

Structural and thermodynamic basis of a frontometaphyseal dysplasia mutation in filamin A

“…2D). This feature is consistent with a molten globule state as documented previously (35). Note that control ANS experiments in denaturant, 6 M guanidine hydrochloride, leads to a loss of these emergent fluorescent features leading to uniform spectral outputs (supplemental Fig.…”

“…Although the broadened NMR peaks may alternatively reflect interchanging fully folded and totally unfolded states on the millisecond time scale, this possibility was eliminated by our subsequent ANS-binding experiments. ANS is known not to bind well to either fully folded or totally unfolded protein (35,36), and this was reflected in our studies ( Fig. 2D and supplemental Fig.…”

“…To further gain insight into the altered folding state of the mutant, we performed 8-anilino-1-naphthalenesulfonic acid (ANS) binding experiment. The enhanced fluorescence from ANS binding to protein is a widely used sensitive marker for differentiating molten globule from folded/unfolded states of proteins (35,36). ANS does not bind appreciably to native or totally unfolded proteins but binds well to partially folded/molten globule state with hydrophobic patches exposed (35,36).…”

“…The enhanced fluorescence from ANS binding to protein is a widely used sensitive marker for differentiating molten globule from folded/unfolded states of proteins (35,36). ANS does not bind appreciably to native or totally unfolded proteins but binds well to partially folded/molten globule state with hydrophobic patches exposed (35,36). We probed ANS binding to Ig20 -21 WT and the P2204L mutant using excitation at 380 nm and recording the emission from 400 -600 nm using a 50 molar excess of ANS (5 M protein and 250 M ANS).…”

Polymorphism on human aromatase affects protein dynamics and substrate binding: spectroscopic evidence.

“…8-Anilino-1-naphthalenesulfonic acid (ANS) is a hydrophobic probe, which increases its fluorescence intensity upon binding to surface cavities and pockets of proteins [20]. For such reason, it is particularly suitable to study the conformational changes that modify the compactness of a protein tridimensional structure, such as those produced by temperature [21], pH [22], pressure [23] and substrate binding [24]. In order to characterize to what extent the presence of substrate (or inhibitor) in the active site of aromatase alter the roughness and accessibility of the protein surface, we added increasing amounts of ANS to the ligand-containing samples and compared the respective titration curves and fit in Figure 6.…”