Fluorescent dyes as probes to study lipid‐binding proteins

Abstract

We studied the equilibrium binding of two hydrophobic fluorescent dyes, ANS and bisANS, to four members of a family of intracellular lipid-binding proteins: IFABP, CRABP I, CRABP II, and ILBP. The spectral and binding parameters for the probes bound to the proteins were determined. Typically, there was a single binding site on each protein for the ligands. However, IFABP cooperatively bound a second bisANS molecule in the binding pocket. Comparative analysis of affinities and spectral characteristics for the two probes allowed us to examine the contributions of electrostatic and hydrophobic interactions to the binding process, and to address some aspects of the internal structure of the studied proteins.

Opening and Closing of the Hydrophobic Cavity of LolA Coupled to Lipoprotein Binding and Release

“…To establish an assay system that can easily distinguish the open/ close structures of LolA and LolA(R43L), we examined the binding of some fluorescent probes and found that the binding of bis-ANS was significantly different between LolA and LolA(R43L). The fluorescence of bis-ANS has been reported to significantly increase when it binds to a hydrophobic environment (16). The fluorescence intensity of bis-ANS was very low in the absence of LolA proteins (Fig.…”

A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography

“…Fluorometric titrations of ANS into L-FABP were performed as previously described [26]. BisANS was used as the binding cavity probe for human I-BABP [27]. The binding properties of PPAR␥LBD were measured by titration with the fluorescent FA cis-parinaric acid as previously described [28].…”

“…The ligand binding affinities of each protein were measured using the fluorescent probes ANS (L-FABP binding measurements), cisparinaric acid (PPAR␥LBD binding measurements) and bisANS (I-BABP binding measurements), that have previously been shown to bind specifically in the lipid binding cavity of these proteins [26][27][28]. The binding data indicated the proteins did not have an appreciable affinity for the fluorescent probes before delipidation, again suggesting endogenous lipids remain protein-bound Fluorescence was normalized as a relative binding, with no-competition (relative binding = 1) and full competition (relative binding = 0).…”

“…The inhibition constant (K i ) for GW1929 binding was calculated according to the equation K i = EC 50 /(1 + [cis-parinaric acid]/K d cis-parinaric acid ). All other data modeling operations were performed as previously described [26][27][28] using GraphPad Prism V4.0 software (GraphPad software, San Diego, CA, USA).…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…The compound 1,8-ANS is a widely employed fluorescent probe that noncovalently attaches to the hydrophobic cavities and/or clefts on protein surfaces and are commonly used to monitor solvent exposed hydrophobic patches as well as protein unfolding mechanism (Cardamone and Puri, 1992;Hawe et al, 2008;Pastukhov and Ropson, 2003;Uversky et al, 1996). fluorescence intensity can also be detected (Hawe et al, 2011).…”