Purification and In Vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides

Randall J. Kimple, Francis S. Willard, David P. Siderovski

Research output: Chapter in Book/Report/Conference proceedingChapter

9 Scopus citations

Abstract

The GoLoco motif is a short polypeptide sequence that binds to heterotrimeric G-protein α subunits of the adenylyl cyclase-inhibitory (Gαio) subclass in a nucleotide-dependent manner (i.e., solely to the GDP-bound ground state). This article describes methods used for the expression, purification, and in vitro evaluation of membrane-permeant tag fusion peptides derived from the GoLoco motif regions of "regulator of G-protein signaling" proteins type 12 (RGS12) and 14 (RGS14) and a consensus GoLoco sequence from the multiple GoLoco motif protein AGS3. Three different fluorescence-based assays are described for evaluating the in vitro function of these GoLoco peptides as guanine nucleotide dissociation inhibitors, including measurements of GTPγS binding and Gα subunit activation by the planar ion aluminum tetrafluoride.

Original languageEnglish
Title of host publicationRegulators of G-Protein Signaling, Part B
PublisherAcademic Press Inc.
Pages419-436
Number of pages18
DOIs
StatePublished - 1 Jan 2004

Publication series

NameMethods in Enzymology
Volume390
ISSN (Print)0076-6879

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    Kimple, R. J., Willard, F. S., & Siderovski, D. P. (2004). Purification and In Vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides. In Regulators of G-Protein Signaling, Part B (pp. 419-436). (Methods in Enzymology; Vol. 390). Academic Press Inc.. https://doi.org/10.1016/S0076-6879(04)90026-2