title: Incorporation of fluorophore-cholesterol conjugates into liposomal and mycobacterial membranes.
abstract: Fluorescently-labeled steroids that emit intense blue light in nonpolar solvent (λem (CH2Cl2)≈440nm, ΦF=0.70) were prepared by treating cholesteryl chloroformate with 4-amino-1,8-naphthalimides. The lipid portion of the conjugates embeds into liposomal membrane bilayers in minutes, leaving the fluorophore exposed to the external aqueous environment. This causes a 40-nm red-shift in λem and significant quenching. DFT optimizations predict the conjugates to be about 30Å long when fully extended, but rotation about the linker group can bring the compounds into an 'L'-shape. Such a conformation would allow the cholesteryl anchor to remain parallel to the acyl chains of a membrane while the fluorescent group resides in the interfacial region, instead of extending beyond it. When incubated with Mycobacterium smegmatis mc2 155, a bacterial species known to use natural cholesterol, the labeled steroids support growth and can be found localized in the membrane fraction of the cells using HPLC. These findings demonstrate stable integration of fluorescent cholesterols into bacterial membranes in vivo, indicating that these compounds may be useful for evaluating cholesterol uptake in prokaryotic organisms.
authors.affiliation: Department of Chemistry, Science and Technology Building, East Carolina University, Greenville, NC 27858-4353, USA; Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA.
authors.forename: Ashley N
authors.initials: AN
authors.lastname: Wercholuk
authors.affiliation: Department of Chemistry, Science and Technology Building, East Carolina University, Greenville, NC 27858-4353, USA; Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA.
authors.forename: Jenna M
authors.initials: JM
authors.lastname: Thuman
authors.affiliation: Department of Chemistry, Science and Technology Building, East Carolina University, Greenville, NC 27858-4353, USA; Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA.
authors.forename: Jordan L
authors.initials: JL
authors.lastname: Stanley
authors.affiliation: Department of Chemistry, Science and Technology Building, East Carolina University, Greenville, NC 27858-4353, USA; Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Sargent
authors.affiliation: Department of Chemistry, Science and Technology Building, East Carolina University, Greenville, NC 27858-4353, USA; Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA.
authors.forename: Eric S
authors.initials: ES
authors.lastname: Anderson
authors.affiliation: Department of Chemistry, Science and Technology Building, East Carolina University, Greenville, NC 27858-4353, USA; Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA. Electronic address: allenwi@ecu.edu.
authors.forename: William E
authors.initials: WE
authors.lastname: Allen
mesh_terms: D001692:Biological Transport; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009161:Mycobacterium / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D002784:Cholesterol
keywords: Cholesterol trafficking; Fluorescence; Membrane transport; Tuberculosis