Purification and in vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides.
The GoLoco motif is a short polypeptide sequence that binds to heterotrimeric G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) 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 GTPgammaS binding and Galpha subunit activation by the planar ion aluminum tetrafluoride.
Department of Pharmacology, Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill 27599, USA.
Randall J
RJ
Kimple
Francis S
FS
Willard
David P
DP
Siderovski
D017607:Aluminum Compounds / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D031541:Fluorescence Resonance Energy Transfer; D005459:Fluorides / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020710:RGS Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence / Q000379:methods
D017607:Aluminum Compounds; D044385:GTP-Binding Protein alpha Subunits; D010455:Peptides; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; C491401:Rgs12 protein, rat; C106374:Rgs14 protein, rat; C050992:tetrafluoroaluminate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005459:Fluorides