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The exogenous introduction of fluorescent lipoproteins into cell membranes is a method for visualizing the cellular traffic of membrane associated proteins, and also for altering the cell surface in a controlled manner. In order to achieve the cell membrane anchoring of proteins and their subsequent fluorescence based detection, a cholesterol derivative was designed. The headgroup of the novel cholesterol anchor contains a fluorescent reporter and a thiol reactive maleimide for protein conjugation. Protein conjugation was demonstrated by the addition of a green fluorescent maleimido anchor to the C-terminus of a Cys extended red fluorescent protein, mCherry. The resulting dual fluorescent cholesteryl lipoprotein was successfully separated from the micellar associates of the surplus fluorescent lipid anchor without denaturing the protein, and the lipoprotein containing only the covalently linked, stoichiometric fluorescent lipid was efficiently delivered to the plasma membrane of live cells. It was demonstrated that the membrane fluorescence could be directly assigned to the protein−anchor conjugate, because no excess of fluorescent lipid species were present during the imaging experiment and the protein and anchor fluorescence colocalized in the cell membrane. Molecular dynamics simulations and subsequent trajectory analysis suggest also the spontaneous and stable membrane association of the cholesterol anchor. Thus, the method could be beneficially applied for studying membrane associated proteins and for preparing mimetics of glycosylphosphatidylinositol (GPI)-anchored proteins to target cholesterol-rich membrane microdomains. ■ INTRODUCTION Lipidation plays an important role in the localization and function of proteins. Four common modifications of proteins with lipid moieties are myristoylation, palmitoylation, prenylation, and the attachment of glycosylphosphatidylinositol (GPI) anchors. The amidation of the protein C-terminus with a GPI glycolipid results in proteins tethered to the extracellular leaflet of the cell membrane. This modification is widespread throughout eukaryotes, and more than 150 human GPIanchored proteins (GPI-APs) are known with various functions: enzymes, receptors, complement regulation proteins, antigens, or adhesion molecules. Beyond the normal physiological functions, GPI-APs are associated with a range of diseases including paroxysmal nocturnal hemoglobinuria, prion diseases, carcinogenesis, and sleeping sickness. The important functional role of the GPI-APs is further evidenced by the embryonic lethality of the GPI-deficient mice. The GPI glycolipids have the conserved Man(α1−2)Man(α1− 6)Man(α1−4)GlcN(α1−6)myoIno glycan core and variations arise in the substitution pattern and in the lipid composition. The lipid part of the mammalian GPIs is phosphatidylinositol with stearyl chains; therefore, the GPIAPs are able to temporarily associate with sphingolipidand cholesterol-rich membrane microdomains, i.e., lipid rafts. This clustering is mainly due to the favorable hydrophobic interactions between the saturated acyl chains of the GPI anchor and the lipid constituents of the rafts. It was evidenced that the lipid raft association of the GPI-APs could be abrogated by cholesterol depletion of the cell membrane and by replacing the GPI moiety with a transmembrane anchor. Received: April 24, 2013 Revised: September 4, 2013 Published: September 10, 2013 Article

Development of fluorescent cholesterol derivatives for the exogenous introduction of proteins to the plasma membrane