Pepanian, Anna, Sommerfeld, Paul ORCID: 0000-0002-8182-0347, Kasprzyk, Renata, Kuehl, Toni, Binbay, F. Ayberk, Hauser, Christoph, Loeser, Reik, Wodtke, Robert, Bednarczyk, Marcelina, Chrominski, Mikolaj, Kowalska, Joanna, Jemielity, Jacek ORCID: 0000-0001-7633-788X, Imhof, Diana and Pietsch, Markus (2022). Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of G alpha i1 Proteins. Anal. Chem., 94 (41). S. 14410 - 14419. WASHINGTON: AMER CHEMICAL SOC. ISSN 1520-6882

Full text not available from this repository.

Abstract

G alpha proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor -mediated intracellular signaling. The role of the G alpha subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and G alpha protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active G alpha i1 protein. An advanced bacterial expression protocol was applied to produce active human G alpha i1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity G alpha i1 binders compared to the commonly used BODIPY FL GTP gamma S. This study thus contributes a new method for future investigations of the characterization of G alpha i and other G alpha protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Pepanian, AnnaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Sommerfeld, PaulUNSPECIFIEDorcid.org/0000-0002-8182-0347UNSPECIFIED
Kasprzyk, RenataUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kuehl, ToniUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Binbay, F. AyberkUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Hauser, ChristophUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Loeser, ReikUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wodtke, RobertUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Bednarczyk, MarcelinaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Chrominski, MikolajUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kowalska, JoannaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Jemielity, JacekUNSPECIFIEDorcid.org/0000-0001-7633-788XUNSPECIFIED
Imhof, DianaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Pietsch, MarkusUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-681555
DOI: 10.1021/acs.analchem.2c03176
Journal or Publication Title: Anal. Chem.
Volume: 94
Number: 41
Page Range: S. 14410 - 14419
Date: 2022
Publisher: AMER CHEMICAL SOC
Place of Publication: WASHINGTON
ISSN: 1520-6882
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
HETEROTRIMERIC G-PROTEINS; GTP-GAMMA-S; ALPHA-SUBUNIT; RAS PROTEINS; PURIFICATION; ACTIVATION; BINDING; INHIBITORS; AFFINITY; EXCHANGEMultiple languages
Chemistry, AnalyticalMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/68155

Downloads

Downloads per month over past year

Altmetric

Export

Actions (login required)

View Item View Item