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1.  A Homogeneous Method to Measure Nucleotide Exchange by α-Subunits of Heterotrimeric G-Proteins Using Fluorescence Polarization 
Abstract
The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the [35S]guanosine 5′-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg2+ concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.
doi:10.1089/adt.2010.0286
PMCID: PMC2957273  PMID: 20662737
2.  High affinity immobilization of proteins using biotin- and GST-based coupling strategies 
Surface Plasmon Resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction.
doi:10.1007/978-1-60761-670-2_4
PMCID: PMC3025018  PMID: 20217614
BIAcore 3000; Biotin; Streptavidin; GST; High-Affinity Immobilization; SPR
3.  Two Gαi1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity 
Journal of biomolecular screening  2009;14(10):1195-1206.
RGS proteins are critical modulators of G protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via “GTPase-accelerating protein” (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow GDP release from Gα and lack of solution-phase assays for detecting free GDP in the presence of excess GTP. To overcome these hurdles, we developed a Gαi1 mutant with increased GDP dissociation and decreased GTP hydrolysis, enabling detection of GAP activity using steady-state GTP hydrolysis. Gαi1(R178M/A326S) GTPase activity was stimulated 6~12 fold by RGS proteins known to act on Gαi subunits, and not affected by those unable to act on Gαi, demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. Gαi1(R178M/A326S) interacted with RGS proteins with expected binding specificity and affinities. To enable non-radioactive, homogenous detection of RGS protein effects on Gαi1(R178M/A326S), we developed a Transcreener® fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gαi1(R178M/A326S) with a homogenous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling.
doi:10.1177/1087057109347473
PMCID: PMC2795102  PMID: 19820068
Fluorescence polarization; GDP detection; regulators of G-protein signaling; surface plasmon resonance

Results 1-3 (3)