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A method is described for the quantitative analysis of protein-protein interactions using the Flow Cytometry Protein Interaction Assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the Regulator of G-Protein Signaling protein, RGS19, in either a saturation or competition format. An adaptation of this method that is compatible for High Throughput screening is also provided.
Nearly all biological processes utilize a protein-protein interaction (PPI) at some level. As our understanding of cellular processes evolves, it has become clear that a thorough understanding of how two or more proteins interact in a functional manner is a critical factor in determining the fundamental mechanisms governing cellular processes. Due to the importance of protein-protein interactions in biology, a number of methods have been developed to study the nature of these binding events in vitro. These methods fall generally into one of three classes: solution-phase methods (e.g. fluorescence polarization, intrinsic fluorescence changes, fluorescence resonance energy transfer (FRET)/fluorescence quenching, isothermal calorimetry, nuclear magnetic resonance (NMR)); cell-based methods (e.g. bioluminescence resonance energy transfer (BRET) reporters, split luciferase reporters, yeast/mammalian two hybrid, beta-galactosidase complementation assays); and solid-surface methods (e.g. co-immunoprecipitation, surface plasmon resonance). Many of these methods are based upon studying the interaction between two or more recombinantly expressed and purified proteins. These approaches, including the methodology being presented here, have proven to be extremely valuable tools in the study of PPIs. The method described here has significant advantages over these more traditional tools, especially for the quantification of PPI affinities and in the development of small molecule protein-protein interaction inhibitors.
This unit describes a method for the quantitative analysis of protein-protein interactions using the flow cytometry protein-protein interaction assay (FCPIA, Figure 1). This methodology is based upon immobilizing a binding partner (target) to polystyrene beads and incubating these beads in a solution of a fluorescently labeled binding partner (reporter) in the presence or absence of competitors. This method can be used to characterize a variety of protein-protein interactions [1-3] and to identify or characterize inhibitors of these binding events [2, 4, 5], even in a high throughput manner (See  and alternative protocol 1). This assay has been designed primarily for use with purified and chemically labeled proteins, but has also been adapted for use with cell lysates and other complex biological mixtures .
The protein-protein interaction upon which most of our work with this protocol is based is that of the interaction between a Regulator of G protein Signaling (RGS) and a heterotrimeric G protein α subunit (Gα). RGS proteins are potent negative modulators of G protein signaling [7-9]. They function by binding to the active (GTP-bound) form of Gα subunits and inducing a conformational change in the G protein that accelerates the rate of GTP hydrolysis . This interaction is heavily dependent upon the nucleotide-bound state of the G protein, whereby the RGS protein binds weakly (Kd 1μM) to the guanosine diphosphate (GDP) state, but binds with high affinity (Kd ~1-10nM) to the GDP-aluminum fluoride state (Figure 2). In this conformation, the aluminum fluoride complex sits in a planar configuration in the gamma phosphate binding site on the G protein. It has been proposed that this conformation is similar in nature to the transition state of GTP hydrolysis . The strong dependence on aluminum fluoride for the RGS-Gα interaction will be used as a means to differentiate between specific and nonspecific binding for this particular PPI.
This assay is applicable to the study many PPIs other than the RGS-Gα interaction. In the nervous system, PDZ domain containing proteins are important scaffolds for signaling systems, especially in the post synaptic density. One particular PDZ domain containing protein, G alpha Interacting Protein C-terminus (GIPC) has been suggested to be important in localizing RGS19 to the D2 and D3, but not D4 dopamine receptor [10, 11]. We have quantified the affinity of GIPC for RGS19 using this method (Figure 3). Furthermore, we show that the affinity is independent of which protein is the “target” protein (e.g. the immobilized protein on the bead). To confirm that the non-stereotypical PDZ motif on the C-terminus of RGS19 (QSSEA) is the predominant motif required for the GIPC-RGS19 interaction, a mutant of RGS19 with a C-terminal truncation of the last 11 amino acids (including the PDZ ligand) was generated. This mutant protein is incapable of competing with wild-type RGS19 for binding to fluorescently labeled GIPC, suggesting that the PDZ motif is indeed necessary for the GIPC-RGS19 interaction. The observation that GIPC and RGS19 interact in a PDZ-dependent manner provides further evidence that GIPC may function as a scaffold for RGS19. For this set of experiments a competition format was particularly useful. Unlike a saturation experiment, the competition experiment directly compares the ability of two proteins to compete for the binding of the reporter protein, as opposed to trying to measure weak or non-existent binding of a PDZ ligand-deficient RGS19 to GIPC.
This assay is also useful for the rapid screening of hybridoma clones in the development of monoclonal antibodies. This method is useful in clone screening because it is easily multiplexed, allowing for the study of the antigenic target and several related targets in a single well. The ability to screen for antigenic specificity earlier in the hybridoma screening reduces both the number of clones for follow up and the time required to identify the optimal clone. To prove this concept, we generated hybridomas from mice that had been immunized with RGS19 and screened these clones for the ability to selectively bind RGS19 over several other related RGS proteins in a multiplexed manner using less than 50 μL of culture supernatant (Figure 4). This was performed by immobilizing RGS proteins or the fusion protein with which the antigen was originally expressed (maltose binding protein; MBP) on the bead. The beads were then incubated with a small volume of hybridoma supernatant and an excess of phycoerythrin (PE) labeled anti-mouse IgG. MBP was included as a control because the RGS19 was expressed and purified as an MBP fusion protein. The MBP tag was proteolytically cleaved from the RGS and purified away before mice were immunized, but there was still the potential for low levels of contamination of the antigen with this bacterial protein.
The FCPIA methodology described in this unit is applicable to the study of many protein-protein interactions. It is amenable to the multiplexing of several target proteins for the binding to a single reporter, saving both time and reagents. The main limitation of this approach is the apparent affinity limitations that we have observed. PPIs with Kd values weaker than 1μM or so have been difficult to resolve using this method.
Avidin coated polystyrene beads are saturated with a purified and biotinylated binding partner and incubated in varying amounts with a purified AlexaFluor-532 labeled binding partner. After equilibration, bead-associated fluorescence is measured by flow cytometry. This application is easily multiplexed by using optically encoded beads, which are commercially available from a variety of sources and a suitable flow cytometer (reviewed in Current Protocols in Cytometry Unit 13.8), allowing for simultaneous detection of reporter protein interactions with multiple targets.
This protocol is designed to measure the saturatable, aluminum fluoride-dependent binding of Gαo to RGS4. This assay requires 32 wells on a 96-well plate, 16 for dilutions of Gαo in the presence of AMF, 16 for dilutions of Gαo in the absence of AMF. The latter provides a measure of non-specific binding and is considered background [4, 12].
It is often desirable to study the inhibition of a PPI by small molecules or competing proteins. This protocol provides a method to determine the inhibitory activity of a competitor (small molecule or protein) on the formation of the Gαo-RGS PPI. This method can be easily modified for single point high-throughput screening ([4, 13], also see Basic Protocol 3).
This protocol is single point version of the competition experiment described in Basic Protocol 2 that has been optimized for high throughput screening. This method has been scaled to 384-well format and, like all protocols described in this unit, is amenable to multiplexing. Using a method very similar to the one presented here, we have screened over 200,000 small molecules using several multiplexed assays to identify inhibitors of Gαo binding to RGS4, RGS19, RGS16, RGS7 and RGS8 both in our laboratory, in collaboration with the Center for Chemical Genomics at the University of Michigan and in collaboration with the Molecular Library Probe Screening Network (MLPCN) at the University of New Mexico ([4, 13] MLPCN screening IDs: 1423, 1415, 1439-41). For simplicity, this assay has been scaled to ten whole 384 well plates. The reader will notice that the fluorescent label on the Gαo has been changed to AlexaFluor-488 for this application. The sole purpose for this was to allow for data acquisition using the standard blue laser available in most cytometers.
Protocol for ten 384-well plates:
This protocol is a general method for biotinylating RGS proteins, although it is generally applicable to most purified protein samples that contain solvent exposed primary amines (e.g. lysine residues or unmodified N-terminus).
Amicon Ultra centrifugal concentrators, 10K MWCO (Millipore Inc., Cat # UFC901096)
This protocol is a general method for fluorescently labeling Gαo, although it is generally applicable to most purified protein samples that contain solvent exposed thiols (e.g. cysteine residues). This protocol is identical for labeling Gαo with AlexaFluor488.
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A, for suppliers, see SUPPLIERS INDEX
Phosphate buffered saline pH 7.4 supplemented with 1% bovine serum albumin (BSA) and sterile filtered through a 0.22μm filter to remove particulates that might be detected in the flow cytometer.
50mM HEPES pH 8.0 at ambient temperature (pH adjusted with 10N NaOH), 100mM NaCl, 0.1% Lubrol PX, supplemented with 1% BSA and sterile filtered through a 0.22μm filter. If the experiment involves Gα subunits, supplement this buffer with 10μM GDP immediately before use. The BSA and detergent concentrations have been optimized to reduce non-specific binding of the RGS-Gα binding pair and may need to be optimized for each PPIs.
A number of methods have been developed to quantitatively assess the binding of two proteins. The FCPIA approach is generally applicable to the study of PPIs. We have tailored this approach to monitor the interaction between a wide array of RGS proteins and Gα subunits, however we and our collaborators have successfully used this method to study the interaction between a variety of different proteins. This methodology is quite malleable and can be adapted to the study of many PPIs. To date, it has been applied to relatively high affinity interactions with Kd values ranging from 100pM to over 300nM. Furthermore, this assay is directly adaptable to high-throughput analysis in a 384 well format. This has been used successfully for small scale HTS [4, 13] and has been scaled up to large HTS screening campaigns (Neubig, unpublished and MLPCN screening IDs: 1423, 1415, 1439-41). This methodology has some particular advantages over standard solution-phase screening methods (e.g. FP, FRET). First and foremost, this method reduces the effect of spectrally interfering compounds or contaminants by virtue of the analysis technique. Also, the large amount of carrier protein (BSA) and detergent that is used in the assay buffer to reduce non-specific binding will also minimize the effects of nonspecific aggregators that have been shown, in certain cases, to constitute a large number of the false positives identified in biochemical screens for small molecule protein-protein interaction inhibitors (SMPPIIs) [14, 15].
Another important advantage is the ability to perform multiplexed binding experiments. In this approach, two or more target proteins are assayed for ability to bind the same reporter protein under conditions of negligible competition between target proteins for free ligand. Thus, multiplexing increases experimental efficiency by reducing reagent/time use and increasing the amount of data generated per experiment. Furthermore, since the binding conditions are truly identical for each target protein, differences in affinity of the targets can be more accurately determined. Using our RGS-Gα interaction example, we have multiplexed up to seven different RGS proteins in a single experiment, although theoretically it should be possible to go significantly higher.
These advantages can be combined to provide a convenient method for high throughput analysis in a multiplexed format. As mentioned previously, we have used this method to perform several high-throughput screens to identify small molecule RGS-Gα inhibitors. In these assays, we screened the library against five different RGS proteins simultaneously, thus reducing time, reagent and compound library use by five-fold.
There are two major limitations to the FCPIA assay. The first limitation is that FCPIA has an affinity limitation that can restrict its use for the characterization of low-affinity interactions. PPI's with low affinities (>1μM Kd) tend to be difficult to resolve in this assay, most likely due to the increased non-specific adsorption of the reporter protein to the bead. This is not a problem with a homogenous solution phase PPI assay, where there is no solid substrate for adsorption. The second major limitation to the FCPIA assay is the potential for non-specific binding that has to be overcome in the assay. For soluble, well-behaved proteins this limitation is not often an issue. For ‘stickier’ proteins this limitation is often circumvented by optimizing conditions with a small detergent/blocking agent (e.g. BSA) screen. Other ways to optimize the signal to noise of particularly troublesome PPI pairs can be found in the next section. Even with these limitations, the ability to dramatically increase data throughput by assay multiplexing makes FCPIA an attractive complement to the more traditional solution phase protein-protein interaction assays.
If the proper precautions are not taken, there can be significant issues with non-specific binding in this assay. As with all biochemical assays, the most specific signal from recombinant proteins will be obtained if the target protein is purified to homogeneity before use. The inclusion of up to 1% BSA in the assay buffer and detergent (e.g. 0.1% Lubrol PX) was able to minimize non-specific binding under most circumstances tested thus far. Determining the optimal concentrations and types of carrier proteins and detergents may be PPI specific. A further mechanism to minimize non-specific fluorescence events in the assay is to ensure that the labeled protein is properly folded and purified away from free label. Biochemical labeling of purified proteins with bulky fluor groups can sometimes cause protein aggregation or misfolding. These improperly folded proteins can contribute significantly to the non-specific binding signal observed. Optimization of the labeling procedure can minimize these effects but it is advisable to purify the labeled protein from the reaction mixture using a size exclusion column to remove both free label and aggregated protein. Other commonly used methods (e.g. spin column, concentration/dilution, dialysis) will not remove aggregated protein and are likely to be less efficient at removing free label.
Low-affinity complexes: The FCPIA approach, unlike some solution-state experiments, has an affinity (Kd) limitation on the order of 1μM. This is likely to be a primarily a product of the nonspecific binding that can be observed.
When performed correctly, this method will provide a robust specific binding signal, often 5-50 fold above background. In many cases, PPIs are high affinity (Kd ~nM) and they will be easily observed in a quantitative manner. Unlike most assays, saturation binding experiments performed in FCPIA are capable of being multiplexed. While in most fluid phase assays the amount of ‘receptor’ protein required for adequate signal detection is at or just under the expected Kd value of the interaction, the FCPIA approach requires significantly less. In the protocols described above, the binding capacity of the beads limits the amount of target protein to the pM-fM range, which is well below the Kd values observed for most PPIs (nM-μM). This provides the advantage that, even at the low concentrations of the reporter protein (e.g. the fluorescently labeled protein), there is minimal competition between different ‘receptor’ proteins in the well. Using this method it is possible to compare the binding of two or more proteins to a ‘ligand’ protein in the same well, saving both time and reagents.
Bead labeling/washing should take approximately 45 minutes. Plate setup and incubation of RGS-labeled beads with fluorescent Gαo should take an additional 40 minutes. Instrument setup and data collection on a full 96-well plate in the Luminex 200™ should take approximately 30 minutes.
Bead labeling/washing should take approximately 45 minutes. Plate setup and incubation of RGS-labeled beads with competitor should take 20 minutes. Then, an additional 30 minutes is required for incubation of the RGS/competitor mixture with fluorescent Gαo. Instrument setup and data collection on a full 96-well plate in the Luminex 200™ should take approximately 30 minutes.
The authors would like to thank Roger Sunahara and John Tesmer at the University of Michigan for helpful discussion. We also thank Martha Larsen of the Center for Chemical Genomics for assistance in the development and application of the high-throughput screening methodology. We would also like to thank Larry Sklar and Yang Wu at the University of New Mexico Center for Molecular Discovery for running the multiplex RGS inhibitor screen. We would like to acknowledge the support of the Michigan Chemistry-Biology Interface Training Program, which is funded through the National Institutes of Health under grant number T32 GM00008597. This work was also supported by GM39561 (RRN) and GM076821 (DLR).