title: 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.
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Black
authors.forename: Will A
authors.initials: WA
authors.lastname: Stanley
authors.forename: Fabian V
authors.initials: FV
authors.lastname: Filipp
authors.forename: Michelle
authors.initials: M
authors.lastname: Bhairo
authors.forename: Ashwani
authors.initials: A
authors.lastname: Verma
authors.forename: Oliver
authors.initials: O
authors.lastname: Wichmann
authors.forename: Michael
authors.initials: M
authors.lastname: Sattler
authors.forename: Matthias
authors.initials: M
authors.lastname: Wilmanns
authors.forename: Carsten
authors.initials: C
authors.lastname: Schultz
mesh_terms: D000478:Alkylation; D000818:Animals; D020148:Butyric Acid / Q000737:chemistry; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry; D002417:Cattle; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D017186:Titrimetry
chemical_list: D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008055:Lipids; D004364:Pharmaceutical Preparations; C072087:sterol carrier proteins; D020148:Butyric Acid; D012710:Serum Albumin, Bovine