Sammenligning affiniteten af ​​GTPase-bindende proteiner under anvendelse af kompetitive assays

Summary

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

Abstract

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

Introduction

The actin cytoskeleton that determines the shape, polarity and migratory properties of mammalian cells is regulated by the Rho-family of small GTPases. The Rho-family GTPases include RhoA that stimulates cytoskeletal contraction, Rac1 that stimulates actin branching and membrane protrusion, and Cdc42 that has similar effects on actin polymerization to Rac1 and causes the formation of filopodia ^1,2. GTPase signaling activity is determined by binding of a nucleotide, which controls the contraction and relaxation of the switch I and switch II loops that mediate the protein-protein interactions with both regulators and effectors. Guanosine 5’-triphosphate (GTP)-bound GTPases activate downstream effectors, whereas the Guanosine 5’-diphosphate (GDP)-bound form is inactive. In the cell, cycles of GTP hydrolysis and nucleotide exchange allow rapid turnover of GTPase signals that are necessary for cytoskeletal dynamics. Nucleotide turnover is regulated by three mechanisms. Guanine nucleotide exchange factors (GEFs) stabilize the nucleotide-free GTPase, catalyzing exchange of GDP for GTP, and thereby stimulating GTPase signaling activity ^3,4. GTPase-activating proteins (GAPs) catalyze hydrolysis of GTP to GDP, thereby inhibiting GTPase signaling activity ⁵. Sequestering molecules such as regulator of chromatin condensation 2 (RCC2) and guanine nucleotide dissociation inhibitors (GDIs) obscure the switch loops and in the case of GDIs remove the GTPase from the membrane by interaction with the prenyl tail ^6,7. Each of the three classes of regulatory molecule interact with the switch loops, as do the downstream effectors and some trafficking regulators such as coronin-1C ⁷. The purpose of this protocol is to measure competition for the switch I/II binding site between putative regulators and downstream signaling molecules. It should be noted that competition assays test binding to a shared binding site, so that this protocol is not suitable for testing interactions with other sites, such as binding of GDIs to the prenyl tail.

The subtlety of the conformation differences between active and inactive forms, combined with the labile nature of the bound nucleotide, has made study of GTPase-binding events difficult. The role of the bound nucleotide means that conventional binding assays such as immunoprecipitation or surface plasmon resonance are not well suited to investigation, as the nucleotide cannot be controlled. This obstacle is compounded by the overlap in the binding sites of GEFs, GAPs, effectors, sequestering molecules and trafficking molecules, which make binding data for a single interaction difficult to interpret in the context of the competition that will occur in the cell. Immunoprecipitation, in particular, is compromised by competition between binding partners, as under certain cellular conditions, one binding partner might be identified at the expense of all others, while under other conditions, another partner might dominate. The dynamic nature of GTPase signaling is essential to GTPase function and must be considered when analyzing the relationships between the binding interactions of different regulators. Indeed, we recently described a pathway that relied heavily on competitive binding. We identified coronin-1C as a trafficking molecule that bound to the switch loops of GDP-Rac1 ⁷. In areas of low GEF activity, trafficking would dominate, removing Rac1 from those regions. However, when Rac1 is delivered to regions of the cell where GEF activity is high, the GEF would outcompete coronin-1C, thereby both activating Rac1 and preventing coronin-1C-mediated removal of Rac1 from that area. The model goes further, because the action of the GEF exchanges bound GDP for GTP, shifting the equilibrium still further from coronin-1C. Consequently, Rac1 activity could be explained entirely in terms of competition and relative affinity.

In this protocol, we describe a method for comparing the relative affinities of different binding partners for small GTPases, using Rac1 as an example. By using a purified protein approach, it is possible to piece together a chain of signaling events by pair wise comparison, in an experiment where the bound nucleotide can be closely controlled.

Protocol

1. Oprensning af GST-mærket GTPase Kultur et E. coli-stamme, såsom BL21 transformeret med pGEX-Rac1 O / N ved 37 ° C, under omrystning ved 220 rpm, i 500 ml autoinduktionen medier (25 mM Na 2 HPO 4, 25 mM KH 2 PO 4, 50 mM NH4CI, 5 mM Na 2 SO 4, 2 mM MgSO4, 2 mM CaCl2, 0,5% glycerol, 0,05% glucose, 0,2% lactose, 5 g trypton, 2,5 g gærekstrakt, 100 ug / ml ampicillin). Harvest bakterier ved centrifugering i 10 minutter ved 10.000 xg, 4 ° C. Resuspender bakteriel pellet i 20 ml proteinekstraktion reagens, 1x proteasehæmmer og inkuberes i 20 minutter ved stuetemperatur med inversion. Præcisere lysatet ved centrifugering ved 40.000 xg i 30 min. Tilsæt 2 ml glutathion magnetiske perler, vasket med phosphatbufret saltvand (PBS: 10 mM Na 2 HPO 4, 1,8 mM KH 2 PO 4, 137 mM NaCl, 2,7 mM KCI). Inkuber i 2 timer, blanding ved inversion ved 4 ° C. Vask protein-loaded perler fire gange med 10 ml PBS under anvendelse af en magnetisk partikel sorteringsanlæg til udfældning af perlerne ved hvert trin. Resuspender protein-loaded perler i 2 ml PBS og opbevares ved -80 ° C i 100 pi alikvoter indtil de skal bruges. 2. Ekspression af GTPase-bindende proteiner Dagen før eksperimentet transficere plasmider, der koder for grønt fluorescerende protein (GFP) -mærket versioner af hver GTPase-protein i en separat 75-cm2 kolbe med HEK293T som følger. Til validering af nukleotid lastning, transficere GFP-mærkede TrioD1 ind i en tredje 75-cm2 kolbe af HEK293T. Fortynd polyethylamine til 1 mg / ml i 100 pi sterilt 150 mM NaCI. Tilføj 27 pi fortyndet polyethylamine til 223 pi reducerede serum medier. Tilføj 12 ug plasmid-DNA til 250 pi reducerede serum medier. Inkuber hvert rør i 2 minutter ved stuetemperatur. </li> Kombiner polyethylamine og DNA blander i et enkelt rør og vortex i 2 min. Inkuber i 15-20 minutter ved stuetemperatur. Udskifte vækstmedium (Dulbeccos Modified Eagle Media, 10% føtalt bovint serum, 2 mM L-glutamin, ingen antibiotika) på 90% sammenflydende HEK293T med 5 ml frisk vækstmedium. Tilsæt kombinerede polyethylamine / DNA-blandingen til kolben og inkuber O / N ved 37 ° C, 5% CO2. 3. Oprensning af GTPase-bindende proteiner Skyl kolber af transficerede celler i PBS og afløb kolbe i 5 minutter, opsugning fri væske. Skrabe celler i 500 pi lysisbuffer (50 mM Tris-HCI (pH 7,8), 1% Nonidet P-40, 1x protease inhibitor) i mikrofugerør. Lyserer celler ved blanding ved inversion ved 4 ° C i 30 minutter. Under lyse, vask to partier af 40 pi GFP-Trap perler tre gange med frisk lysisbuffer, sedimenterende perler på 2.700 xg i 2 min mellem vaskene. Præcisere lysater ved centrifugering ved 21.000 x g i 10 min. Overførsel klaret lysat af hver af de konkurrerende proteiner at adskille vasket GFP-trap perler og tillader GFP-fusionsproteiner til at binde i 2 timer, blanding ved inversion ved 4 ° C. Hold lysat fra GFP-TrioD1 celler på is. Vask indlæst GFP-Trap perler to gange i 50 mM Tris-HCI (pH 7,8), 50 mM NaCl, 0,7% (vægt / volumen) Nonidet P-40 og to gange i 50 mM Tris-HCI (pH 7,6), 20 mM MgCl2 , sedimenterende perler ved 2.700 xg i 2 minutter mellem vaske. Eluering GFP-fusionsproteiner ved at tilsætte 40 pi 0,2 M glycin (pH 2,5) og pipettere op og ned i 30 sek. Straks sediment perler på 21.000 xg i 60 s og flydende overførsel til et nyt mikrocentrifugerør indeholdende 4 pi 1 M Tris-HCl (pH 10,4). Gør dette hurtigt for at begrænse skader på det rensede protein. Analyser 1 pi af hvert oprenset protein ved Western blot og sonde med et anti-GFP-antistof til at etablere relative udbytte under anvendelse af en kvantitativ Blotting ifølge producentens protokol. Alternativt, fastlægge proteinkoncentrationer af bicinchoninsyre (BCA) assay men dette indfører fejl, hvis proteinerne ikke reagerer med assayet, på samme måde, eller der er kontaminerende proteiner. Udligne molær proteinkoncentration ved tilsætning af 50 mM Tris-HCI (pH 7,6), 20 mM MgCl2. 4. Nucleotide belastning af GTPase Tø en portion af GST-Rac1 magnetiske perler, fremstillet i Trin 1. Tag 90 pi GST-Rac1 perler og vaskes tre gange med 20 mM Tris-HCI (pH 7,6), 25 mM NaCl, 0,1 mM DTT, 4 mM EDTA, ved hjælp af en magnetisk partikel sorteringsanlæg til udfældning af perlerne ved hvert trin. Aspirer buffer fra perler og tilsættes 100 pi 20 mM Tris-HCI (pH 7,6), 25 mM NaCl, 0,1 mM DTT, 4 mM EDTA. Efter om BNP, GTP eller ingen nukleotid læsning er nødvendig for konkurrencen eksperimentet, tilsættes 12 pi 100 mM BNP, 12 pi 10 mM guanosine 5 '- [γ-thio] triphosphat (GTPyS) eller ingen nukleotid til 60 pi GST-Rac1 perler. For nukleotid-loading kontroller, opdele de resterende perler i tre 10-pi prøver og tilsæt 2 pi 100 mM BNP, 2 pi 10 mM GTPyS eller ingen nukleotid til hvert rør. Inkuber perle blandinger i 30 minutter ved 30 ° C under omrøring. Stabiliser nukleotid-bundet Rac1 ved tilsætning af 1 M MgCl2: 3 pi til den eksperimentelle mix (trin 4.4), 0,5 pi til hvert kontrolpunkt blandinger (trin 4.5). 5. Konkurrence binding. Opsætning 6 mikrocentrifugerør, der hver indeholder: 200 pi 50 mM Tris-HCI (pH 7,6), 20 mM MgCl2 10 pi eksperimentelle nucleotid-loaded Rac1 perler (fra trin 4.7) 5 pi Rac1-bindende protein A (konstant bindingsprotein) Til hvert rør tilsættes 0, 1, 2,5, 5, 10 eller 20 pi Rac1-bindende protein B (variabel bindende protein). Disse mængder påtage sig enpproximately måske skal justeres lige lager koncentrationer af de konstante og variable bindende proteiner og. Juster volumener bindingsproteiner A og B, hvis der er store forskelle i bindingsaffiniteter for de to proteiner, og dette bør bestemmes empirisk ved de eksperimentelle gentagelser. Der fyldes op til totale volumen af bindingsblanding til 235 pi ved tilsætning af 50 mM Tris-HCI (pH 7,6), 20 mM MgCl2. Opsæt en mikrocentrifugerør indeholdende: 200 pi 50 mM Tris-HCI (pH 7,6), 20 mM MgCl2 10 pi eksperimentelle nucleotid-loaded Rac1 perler (fra trin 4.7) 10 pi Rac1-bindende protein A (konstant bindingsprotein) Opsæt BNP, GTPyS og ingen nukleotid kontrol rør: 200 pi 50 mM Tris-HCI (pH 7,6), 20 mM MgCl2 10 pi kontrol Rac1 perler er lagt i trin 4.5 med BNP, GTPyS eller ingen nukleotid og stabiliseret i trin 4.7. 180 pi HEK293T GFP-TrioD1 lysat, fremstillet som i trin 3.6 4 pi 1 M MgCl2 Inkuber blandingen i 2 timer, blanding ved inversion ved 4 ° C. Vask perlerne tre gange med 50 mM Tris-HCI (pH 7,6), 20 mM MgCl2. Eluerer bundne proteiner i 20 pi reducerende prøvebuffer (50 mM Tris-HCl (pH 7), 5% SDS, 20% glycerol, 0,02 mg / ml bromphenolblåt, 5% β-mercaptoethanol). 6. Analyse af konkurrencen Løse 10 pi af det bundne protein (trin 5.6) ved hjælp af natriumdodecylsulfat-polyacrylamidgelelektroforese (SDS-PAGE) og Western blot. Inkuber membranen ved 4 ° CO / N i anti-GFP-antistof fortyndet 1/1000 i blokerende buffer fortyndet til 1x i PBS, 0,1% Tween-20 til at detektere begge de mærkede GTPase-bindende proteiner. Vask membranen tre gange i 10 minutter med PBS, 0,1% Tween-20. Inkuber membranen i 30 minutter ved stuetemperatur i DyLight 800-konjugeret anti-kanin-secdært antistof, fortyndet 1 / 10.000 i blokerende buffer fortyndet til 1x i PBS, 0,1% Tween-20. Vask membranen tre gange i 10 minutter med PBS, 0,1% Tween-20. Scan membranen ved hjælp af en infrarød billeddannelse system, ved hjælp af software til at måle båndintensitet ifølge producentens protokol. Plot båndet intensiteten af ​​hvert protein mod mængden af ​​den variable konkurrent (Protein B). Opdele mængden af ​​variable konkurrent på det sted, hvor de krydser hinanden ved omfanget af konstant konkurrent (Protein A, 5 pi) til bestemmelse af forholdet konkurrent ved hvilken ligevægt opnås. Til validering af nukleotid-lastning status, probe membraner til p21-aktiveret kinase 1 (Pak1) (en effektor) og GFP-TrioD1 (en GEF), som beskrevet i trin 6.1-6.6.

Representative Results

Denne protokol er udviklet til at beregne de relative affiniteter af bindingspartnere for Rac1, uden behov for at kende den præcise koncentration af konkurrenterne (figur 1). Bestemmelse af proteinkoncentration introducerer fejl og ved vurdering af konkurrencen mellem molekyler i en signalvej er ikke nødvendigt. Det er imidlertid vigtigt at vide, at de to konkurrenter har den samme molære koncentration i stamopløsningerne at tillade simple forhold, der skal beregnes, når tilsætning af forskellige mængder til analysen. 40 pi GFP-Trap perlerne har en bindingskapacitet ~ 300 pmol så en sammenflydende 75 cm 2 kolber af stærkt udtrykkende celler vil mætte perlerne, med det resultat, at præparater af de to forskellige bindingsproteiner vil være de samme før justering (figur 2A). Hvis et af proteinerne udtrykker dårligt, kan dette problem overvindes ved at oprense det protein fra mere end én kolbe af celler. <p class = "jove_content"> Bindingen af ​​de fleste GTPase effektorer og regulatorer afhænger af nukleotid-belastning af agn GTPase, så det er vigtigt at teste, om lastning har været en succes. Loading kan verificeres ved bundfældning kendte bindende proteiner fra cellelysater. Effektor proteiner, såsom Pak1 binder til GTP-Rac1 og kan nemt udfældes fra lysater og påvist ved Western blotting 8 (figur 2B). GEFs fortrinsvis binder til nukleotid-fri GTPase at stabilisere overgangen tilstand. Som GEFs er af lav tæthed, som regel inaktiv og ofte skamplet dårligt, er det bedre at overudtrykke en eller GEF GEF fragment til test nukleotid-fri GTPase. Vi bruger ofte det første Dbl homologi af Trio, udtrykt som en GFP-fusion (GFP-TrioD1 9) (Figur 2B), men enhver GEF ville arbejde. Proteiner, der binder til BNP-loaded GTPase er sjældnere. Vi har for nylig rapporteret RCC2 som ét sådant protein 7, eller BNP-belastning kan valideres blot som binding til hverken GEF eller effektor. Outputtet fra forsøget vil være en Western blot, der afbilder de to GFP-mærkede bindingspartnere bundet til GTPase. Ved at anvende en enkelt antistof til påvisning begge proteiner, kan de koncentrationer, hvor tilsvarende mængder af både konkurrenter binder bestemmes og derfor relative affiniteter udledes. I dette eksempel konkurrence mellem propellen domæne i proteinet Rac1-trafficking, er coronin-1C (Rac1-bindende protein A), og Rac1-kompleksdannende protein, RCC2 (Rac1-bindende protein B), påvist (figur 3A). Ved at bruge en konstant volumen af ​​coronin-1C propel (5 pi), og tilføje stigende mængder RCC2, kan vi se fra GFP skamplet, at ligevægten nås ved 1,25-2,5 ul RCC2 (stjerne), hvilket viser, at RCC2 har en stærkere affinitet for Rac1 end coronin-1C. Ved at måle intensiteten af ​​bånd ved hjælp af kvantitative Western blotting og plotte middelværdier for hver competitor kan ligevægtspunktet beregnes nøjagtigt ved at identificere de mængder, hvor kurverne krydser hinanden (figur 3B). En af de mulige hindringer for en vellykket assay er, hvis de bindende partnere binder til hinanden samt binding til Rac1. I figur 3A + B demonstrerer vi konkurrencen mellem RCC2 og propellen domæne af coronin-1C, snarere end fuld længde coronin-1C. Grunden til at det afkortede coronin er, at coronin-1C også binder RCC2 gennem halen domæne. Når fuld længde coronin-1C titreres mod RCC2, påvises binding af begge proteiner, som følge af ternære kompleksdannelse, frem for konkurrence (figur 3C). Hvis der sker konkurrencen, vil bindingen af ​​ét protein stige, mens de andre falder, og total bundet GFP-fusion vil være konstant. I tilfælde, hvor et ternært kompleks dannes det er nødvendigt at trunkere en af ​​GTPase-protein, således at COMPETkondensatorerne ikke længere interagerer. Figur 1. Workflow. Skematisk fremstilling af arbejdsgangen til bestemmelse af affiniteten af GTPase proteiner ved hjælp af konkurrencemæssige analyser. Klik her for at se en større version af dette tal. Figur 2. Validering af oprensede proteiner. (A) Oprenset GFP-mærkede Rac1 bindingsproteiner analyseret ved Western blot, probing med anti-GFP at bestemme den relative udbytte af de to proteiner. Denne type udligning under eksperimentet tillader koncentrationen af de to proteiner, der skal justeres, så de passer i bindende eksperiment. (B) GDP, GTPyS og ingen nukleotid-loaded GST-Rac1 blev inkuberet med lysatet fra HEK293T udtrykker GFP-TrioD1 og proteiner detekteret ved at plotte for endogen Pak1 eller overudtrykt GFP-TrioD1. Klik her for at se en større version af dette tal. Figur 3. Western blot analyse af relativ proteinbinding. Eksempel udgange fra konkurrencemyndighederne-bindende assays. (A) BNP-loaded Rac1 blev blandet med 5 pi GFP-coronin-1C propel domæne og stigende mængder af GFP-RCC2 blev titreret i. Ved Western blotting bundne proteiner for GFP, er problemer med forskellen påvisning af de to proteiner undgås, og GFP-signalet rapporterer molforholdet mellem de to fusionsproteiner. Stjerner angiver de konkurrencemæssige forhold på eitheR side af ligevægtspunktet. (B) båndintensiteter af bundne GFP fusionsproteiner fra tre uafhængige eksperimenter blev målt ved kvantitativ Western blotting under anvendelse af fluorofor-konjugerede sekundære antistoffer og gennemsnit planlagt at beregne mængden af RCC2 nødvendige for at nå ligevægt. (C ) Eksempel output fra et eksperiment, hvor Rac1-bindende proteiner binder til hinanden og danner et ternært kompleks, i stedet for at konkurrere. BNP-loaded Rac1 blev blandet med 5 pi GFP-RCC2 og stigende mængder af GFP-coronin-1C fuld længde blev titreret i. Stigningen i bundet GFP-coronin-1C uden tab af bundet GFP-RCC2 indikerer ternære kompleksdannelse. Venligst klik her for at se en større version af dette tal.

Discussion

This protocol describes a method for comparing the relative affinities of pairs of small GTPase-binding proteins. The key steps are the preparation of purified GTPase-binding proteins and the nucleotide loading of the GTPase. The use of GTPase-binding proteins with the same GFP tag, allows the concentrations at which similar amounts of each competitor binds to be accurately determined. The use of recombinant nucleotide-loaded GTPase allows interrogation of the binding properties of the GTPase under specific activity conditions. This step is also the most sensitive as nucleotides will both hydrolyze and detach from the GTPase if the magnesium conditions are not maintained precisely.

In the cell, the large number of GTPase-binding proteins combined with the rapid nucleotide turnover makes such pathways difficult to interpret. The simplicity of this method in comparing only pairs of binding proteins and using carefully controlled nucleotide-loading conditions allows signaling pathways to be elucidated. However, the greatest strength of the protocol is also the greatest weakness as it is a simplification of the in vivo situation. Competition assays can be used to build a robust hypothesis, but this should then be tested in cells by knockdown experiments.

There are three features that must be considered when selecting the GFP-tagged GTPase-binding proteins to be used in the experiment. First, the fusion proteins must express well in mammalian cells, such as HEK293T, as competition assays require a reasonable amount of protein. Second, it must be possible to purify the recombinant protein without significant degradation, and where this is not possible, cloning of a GTPase-binding fragment should be considered. Third, the two GTPase-binding proteins must resolve from one another on SDS-PAGE to allow analysis in section 6.

There are a number of potential caveats to the experiment that need to be considered, and possibly addressed:

Possible denaturation of purified GTPase-binding proteins during the acid elution step or steric hindrance by the GFP tag. In our hands, these have not been a problem, but must be tested. The purified proteins can be tested in functional assays ¹⁰. Commercial kits now exist for testing the activity of GEFs or GAPs without the need for isotope-labeled nucleotides. Sequestering proteins, by their nature protect GTPases from GEF or GAP activity, so can be used as competitive inhibitors in the commercial GEF or GAP assays, as we did in our recent publication ⁷. The relevant feature of proteins that traffic GTPase are the capacity to bind the GTPase, and this can be tested easily in a pull down assay. An alternative approach to testing protein integrity that is applicable to all binding proteins is to titrate protein eluted from GFP-trap beads with glycine with the same protein removed from GFP-trap beads by enzymatic cleavage. The experiment would be analyzed by probing both the GFP-tagged and cleaved protein with an antibody against the protein itself. If the protein is undamaged by elution, equilibrium should be achieved at a 1:1 ratio. This approach would also indicate whether the presence of the GFP tag itself compromises the binding properties of the candidate protein, though this does require the production of a construct with an enzymatic cleavage site between the tag and the binding protein. Whether the protein is compromised by the tag or the elution step, the problem could be addressed by modifying the protocol to use an alternative purification method. Rather than GFP, binding proteins could be His-tagged, purified using Ni-NTA and analyzed using an antibody against the His-tag. The important feature is that both binding proteins must share a common tag although, if necessary, two tags could be added to a protein, one for purification and the other for detection.

The protocol is designed to investigate competition between interactions with the switch I/II domains. Although the majority of GTPase interactions are mediated by this motif, there are some exceptions, most notably the interactions of GDIs that bind to the prenyl tail, as well as obscuring the switch domains. In principle, the protocol could be adapted to use GTPase purified from mammalian cells, so that the GTPase is prenylated, however, the presence of multiple binding sites or allosteric effects complicate the interpretation of competition-binding data. Further problems associated with such a modification are that GDIs co-purify with GTPase from mammalian cells, compromising the purity of the isolated proteins and the hydrophobic nature of the prenyl groups means that prenylated GTPases are associated with either GDI or lipid membrane and such factors would need to be considered in the experiment.

The amount of GST-Rac1 being used in the assay. The constant GTPase binding protein must be at a greater concentration than the Rac1, or when the competitor is added, it will simply bind to free Rac1. It will be immediately obvious if this has happened as binding of the competitor, without a loss of the constant protein, will be detected in much the same way as when the two competing proteins bind to one another as shown in Figure 3B. As an additional control (Step 5.3), a binding reaction containing double the amount of constant binding protein and no variable binding protein should be included (Step 5.3). If the Rac1 in the titration experiment is saturated, doubling the amount of constant binding protein will have no effect on the output. The volumes suggested in the protocol should be appropriate, but the amount of Rac1 can be easily reduced. If binding of the competitor without loss of the constant binding partner is observed, reducing the amount of Rac1 should be attempted before trying to map binding sites to avoid ternary complex formation.

Non-specific interaction of GTPase-binding proteins with the GST or bead, as well as specifically with Rac1. This problem would be manifested by residual binding of the constant GTPase-binding protein, even when the variable GTPase-binding protein has reached a plateau at high concentration. Identification of this issue will be aided by conducting reciprocal experiments where the constant and variable GTPase-binding proteins are swapped. Reciprocal experiments will also greatly improve the accuracy of the estimate of equilibrium point, so should always be included. In cases of non-specific binding, the relative concentrations at which equilibrium is achieved can still be calculated by comparing band intensity between the maxima and minima for each protein, or by measuring the extent of non-specific binding by using GST beads as bait, rather than GST-Rac1.

Pull down assays using different nucleotide-loading conditions should be used to complement the competition assay described in this protocol. Determining the nucleotide preference of partners is important for both understanding the competition events and understanding the signaling pathway that the GTPase-binding protein is involved in. In Figure 2B we analyze binding of proteins with established preference for GTP-loaded or nucleotide-free GTPase as a means to validate nucleotide loading. However, it is sensible to investigate the effect of nucleotide loading on each of the competitors as well. If the hypothetical competitors show different preferences, competition will make less of a contribution to the signaling pathway, and indeed nucleotide turnover is likely to be the mechanism that directs exchange of the binding proteins.

Materials

Bugbuster	Novagen	70584-3
COMPLETE protease inhibitor	Roche	05 056 489 001
Glutathione magnetic beads	Pierce	88821
Polyethylenimine, branched, average Mw ~25,000	Sigma Aldrich	408727-100ML
OPIMEM	Life Technologies	31985-047
Dulbecco's Modified Eagle Media	Sigma Aldrich	D5796
Fetal Bovine Serum	Life Technologies	10270-1-6
L-Glutamine	Life Technologies	25030-024
GFP-Trap_A	Chromotec	gta-20
GDP	Sigma Aldrich	G7127	Highly unstable. Aliquot and store at -80 immediately upon reconstritution
GTPγS	Sigma Aldrich	G8634	Highly unstable. Aliquot and store at -80 immediately upon reconstritution
Blocking Buffer	Sigma Aldrich	B6429
Tween-20	Sigma Aldrich	P9416
Anti-GFP antibody	Living Colors	632592	Use at 1/1000 dilution
DyLight 800 conjugated goat anti-rabbit secondary antibody	Fisher Scientific	10733944
Anti-PAK1 antibody	Cell Signaling	2602S	Use at 1/1000 dilution
Odyssey SA Infrared Imaging System	Li-cor	9260-11PC