A Method to Generate and Analyze Modified Myristoylated Proteins

Huanyao Gao, Wei Sun, Zhiquan Song, Yanbao Yu, Li Wang, Xian Chen, Qisheng Zhang

Research output: Research - peer-reviewArticle

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.

LanguageEnglish (US)
Pages324-330
Number of pages7
JournalChemBioChem
Volume18
Issue number3
DOIs
StatePublished - Feb 1 2017

Fingerprint

Proteins
ADP-Ribosylation Factor 1
Lipids
hydrazine
Monomeric GTP-Binding Proteins
Fluorophores
Guanosine Triphosphate
Ketones
Recombinant Proteins
Liposomes
Mass spectrometry
Fluorescence
Imaging techniques
Optical Imaging
Mass Spectrometry

Keywords

  • ADP-ribosylation factor
  • membrane proteins
  • myristoylation
  • protein modifications
  • small GTPase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

Cite this

A Method to Generate and Analyze Modified Myristoylated Proteins. / Gao, Huanyao; Sun, Wei; Song, Zhiquan; Yu, Yanbao; Wang, Li; Chen, Xian; Zhang, Qisheng.

In: ChemBioChem, Vol. 18, No. 3, 01.02.2017, p. 324-330.

Research output: Research - peer-reviewArticle

Gao, H, Sun, W, Song, Z, Yu, Y, Wang, L, Chen, X & Zhang, Q 2017, 'A Method to Generate and Analyze Modified Myristoylated Proteins' ChemBioChem, vol 18, no. 3, pp. 324-330. DOI: 10.1002/cbic.201600608
Gao H, Sun W, Song Z, Yu Y, Wang L, Chen X et al. A Method to Generate and Analyze Modified Myristoylated Proteins. ChemBioChem. 2017 Feb 1;18(3):324-330. Available from, DOI: 10.1002/cbic.201600608
Gao, Huanyao ; Sun, Wei ; Song, Zhiquan ; Yu, Yanbao ; Wang, Li ; Chen, Xian ; Zhang, Qisheng. / A Method to Generate and Analyze Modified Myristoylated Proteins. In: ChemBioChem. 2017 ; Vol. 18, No. 3. pp. 324-330
@article{11ad954ceab445c09fdc617d3d6a5510,
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.",
keywords = "ADP-ribosylation factor, membrane proteins, myristoylation, protein modifications, small GTPase",
author = "Huanyao Gao and Wei Sun and Zhiquan Song and Yanbao Yu and Li Wang and Xian Chen and Qisheng Zhang",
year = "2017",
month = "2",
doi = "10.1002/cbic.201600608",
volume = "18",
pages = "324--330",
journal = "ChemBioChem",
issn = "1439-4227",
publisher = "Wiley-VCH Verlag",
number = "3",

}

TY - JOUR

T1 - A Method to Generate and Analyze Modified Myristoylated Proteins

AU - Gao,Huanyao

AU - Sun,Wei

AU - Song,Zhiquan

AU - Yu,Yanbao

AU - Wang,Li

AU - Chen,Xian

AU - Zhang,Qisheng

PY - 2017/2/1

Y1 - 2017/2/1

N2 - 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.

AB - 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.

KW - ADP-ribosylation factor

KW - membrane proteins

KW - myristoylation

KW - protein modifications

KW - small GTPase

UR - http://www.scopus.com/inward/record.url?scp=85007595741&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85007595741&partnerID=8YFLogxK

U2 - 10.1002/cbic.201600608

DO - 10.1002/cbic.201600608

M3 - Article

VL - 18

SP - 324

EP - 330

JO - ChemBioChem

T2 - ChemBioChem

JF - ChemBioChem

SN - 1439-4227

IS - 3

ER -