Probing lipid- and drug-binding domains with fluorescent dyes

Abstract

A series of 2-and 3-OH Nile red dyes was prepared in order to generate water-soluble probes that could be used to probe lipid binding to proteins. Various substitutions in positions 2-/3-, 6-, and 7-shifted wavelengths while maintaining the environmental sensitivity of Nile red. In order to increase the solubility of the dyes in aqueous solutions, we attached butyric acid groups to the 2-or 3-OH position. In addition, phenothiazine dyes, which exhibited particularly long excitation properties, were synthesized and tested for the first time. All dyes showed Stoke's shifts of 70-100 nm and changes in excitation and emission of over 100 nm, depending on the hydrophobicity of the environment. Binding studies with bovine serum albumin and the non-specific lipid transfer protein SCP2 revealed emission changes of more than 30 nm upon binding to the protein and a five-fold increase in emission intensity. Titration of the dye-loaded proteins with various lipids or drugs replaced the dye and thereby reversed the shift in wavelength intensity. This allowed us to estimate the lipid binding affinity of the investigated proteins. For SCP2, isothermal calorimetry (ITC) data verified the titration experiments. NMR titration experiments of SCP2 with Nile red 2-O-butyric acid (1a) revealed that the dye is bound within the lipid binding pocket and competes with lipid ligands for this binding site. These results give valuable insight into lipid and drug transport by proteins outside and inside cells.

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

“…1a). These assays demonstrated tight binding of a set of LCFA-CoAs, concurring with published data [14][15][16][17]. However, a set of LCFA-carnitines-a pool of which are present in the peroxisomal matrix [21,22]-were found not to bind to any SCP2 variant.…”

“…The synthesis, fluorescent properties and use in ligand competition assays of NR-BA are described in [17]. In brief, samples of SCP2 variants were prepared at 8 lM in 100 mM potassium phosphate (pH 7.0) and preincubated with 250 nM NR-BA for 30 min.…”

“…To verify the data obtained from the ESI-MS assay in an aqueous environment an indirect assay was employed, utilizing NR-BA as an environmentally sensitive competitive ligand for SCP2 [17]. Exposure of SCP2, preincubated with 250 nM NR-BA, to excitation at 540 nm produces a strong fluorescence maximum at 618 nm.…”

“…We have employed a novel and direct in vitro assay using ''soft'' or nearly native electrospray ionisation mass spectrometry (ESI-MS) and verified the results with a competitive binding assay, using a derivative of Nile Red, Nile Red butyric acid (NR-BA, [17]). We have examined LCFA-CoA binding to four variant forms of human SCP2: pre-and mSCP2, each in the presence and absence of the C-terminal PTS1 tripeptide (Fig.…”

“…Amongst the numerous ligands of SCP2 are LCFAs (discussed extensively in [10,11] with structural analysis in [12,13]) and LCFA-CoAs [14][15][16][17][18] as well as a number of intermediates of LCFA b-oxidation-a primary function of the peroxisome [16]. In addition to the high affinity for these ligands, the observed direct association of peroxisomal SCP2 with b-oxidation enzymes acyl CoA oxidase, thiolase A and MFE-2 [19] has led to the suggestion that a function of SCP2 is to stabilise LCFA b-oxidation intermediates and to present them to the appropriate enzymes for further processing (reviewed in [20]).…”