A Method to Generate and Analyze Modified Myristoylated Proteins. (3rd January 2017)

• Record Type: Journal Article
• Authors: Gao, Huanyao; Sun, Wei; Song, Zhiquan; Yu, Yanbao; Wang, Li; Chen, Xian; Zhang, Qisheng
• Is Part Of: Chembiochem. Volume 18:Number 3(2017)
• Page Start: 324
• Page End: 330
• Publication Date: 2017-01-03
• Subjects: ADP-ribosylation factor -- membrane proteins -- myristoylation -- protein modifications -- small GTPase; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201600608
• Languages: English
• ISSNs: 1439-4227

Abstract

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. Abstract : Decoding the myristoylation mystery : Engineered lipid motifs have been used to identify and modify naturally lipidated proteins, but the characterization of the modified proteins was limited by methods to generate and analyze them. 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).