title: A Method to Generate and Analyze Modified Myristoylated Proteins.
abstract: Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio-orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone-modified myristoylated Arf1 could be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems.
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC, 27599, USA.
authors.forename: Huanyao
authors.initials: H
authors.lastname: Gao
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC, 27599, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC, 27599, USA.
authors.forename: Zhiquan
authors.initials: Z
authors.lastname: Song
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, 120 Mason Farm Road, Chapel Hill, NC, 27599, USA.
authors.forename: Yanbao
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, 120 Mason Farm Road, Chapel Hill, NC, 27599, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, 120 Mason Farm Road, Chapel Hill, NC, 27599, USA.
authors.forename: Xian
authors.initials: X
authors.lastname: Chen
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC, 27599, USA.
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D006868:Hydrolysis; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D019814:Myristic Acid / Q000737:chemistry*; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry
chemical_list: D005456:Fluorescent Dyes; D006834:Hydrazines; D008081:Liposomes; D010455:Peptides; D011994:Recombinant Proteins; D019814:Myristic Acid; C029424:hydrazine; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1
keywords: ADP-ribosylation factor; membrane proteins; myristoylation; protein modifications; small GTPase