Preparation of Semisynthetic Lipoproteins with Fluorescent Cholesterol Anchor and Their Introduction to the Cell Membrane with Minimal Disruption of the Membrane

详细信息    查看全文

• 作者：Bal谩zs Sch盲fer ; Erika Orb谩n ; Attila Borics ; Krisztina Husz谩r ; Antal Nyeste ; Ervin Welker ; Csaba T枚mb枚ly
• 刊名：Bioconjugate Chemistry
• 出版年：2013
• 出版时间：October 16, 2013
• 年：2013
• 卷：24
• 期：10
• 页码：1684-1697
• 全文大小：581K
• 年卷期：v.24,no.10(October 16, 2013)
• ISSN：1520-4812

文摘

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鈥揳nchor 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.