Name: comparing the affinity of gtpase-binding proteins using competition assays
Uploaded: 2015-10-08T15:00:00
Duration: 10 min 37 s
Description: Watch this Scientific Journal Video about Comparing the Affinity of GTPase-binding Proteins using Competition Assays at JoVE.com

This work was supported by Wellcome Trust grant 088419 to MDB.

GST標識GTPアーゼの1精製<ol><li>文化E.そのような220rpmで振盪しながら37℃でのpGEX-Rac1のO / Nで形質転換されたBL21として大腸菌株 、自己誘導培地500ml中（中の25mMのNa 2 HPO 4、25mMのKH 2 PO 4、50mMのNH 4 Clを 、 5ミリモルの Na 2 SO 4、2mMのMgSO 4を、2ミリモルのCaCl 2、0.5％グリセロール、0.05％グルコース、0.2％のラクトース、5 gでトリプトン、2.5グラムの酵母エキス、100μg/ mlのアンピシリン）。 </li><li>万XG、4℃で10分間の遠心分離によって収穫細菌。 </li><li> 20 mlのタンパク質抽出試薬中に細菌ペレットを再懸濁し、1×プロテアーゼ阻害剤および逆に室温で20分間インキュベートします。 </li><li> 30分間40,000×gで遠心分離することによって、溶解物を明確にします。 </li><li>リン酸緩衝生理食塩水で洗浄し、2 mlのグルタチオン磁気ビーズ、追加（PBS：10mMののNa 2 HPO 4、1.8mMのKH 2 PO 4、137mMの塩化ナトリウム、2.7mMの塩化カリウム）。 </li><li> 4℃での反転により混合、2時間インキュベートします。 </li><li>各ステップで、ビーズを沈殿させ、磁性粒子ソーターを使用して、10 mlのPBSでタンパク質を負荷したビーズを4回洗浄します。 </li><li>必要になるまで再懸濁し、100μlのアリコートで-80℃で2mlのPBS中のビーズと店をタンパク質ロードされています。 </li></ol> GTPアーゼ結合タンパク質の2発現<ol><li>次のように一日の実験の前に、緑色蛍光タンパク質（GFP）をコードするプラスミドをトランスフェクションHEK293Tの別個の75-cm 2のフラスコにそれぞれのGTPアーゼ結合タンパク質のバージョン-タグ。ヌクレオチド負荷の検証のために、HEK293Tの第75-cm 2のフラスコにGFPタグTrioD1をトランスフェクション。 <ol><li> 100μl中の1mg / mlの滅菌150mMのNaClにポリエチルアミンを希釈します。 </li><li> 223μlの減少血清培地に27μlの希釈したポリエチルアミンを追加します。 </li><li> 250μlの減少血清培地に12μgのプラスミドDNAを追加します。 </li><li>室温で2分間、各チューブをインキュベートします。 </LI&gt; <li>ポリエチルアミンを結合し、DNAが2分間の単一の管、ボルテックスで混合します。 </li><li>室温で15〜20分間インキュベートします。 </li><li> 5ミリリットルの新鮮な増殖培地と90％のコンフルエントHEK293Tに増殖培地（ダルベッコ改変イーグル培地、10％ウシ胎児血清、2mM L-グルタミン、抗生物質なし）を交換してください。 </li><li>フラスコに合わせたポリエチルアミン/ DNA混合物を追加し、37℃、5％CO 2でO / Nインキュベートします。 </li></ol></li></ol> GTPアーゼ結合タンパク質の精製3. <ol><li> PBS中でトランスフェクトされた細胞のフラスコをすすぎ、5分間フラスコをドレイン、自由な液体を吸引します。 </li><li>マイクロチューブに500μlの溶解バッファー（50 mMトリス - 塩酸（pH7.8）、1％のNonidet P-40、1×プロテアーゼ阻害剤）で細胞を削り取ります。 </li><li> 4℃で30分間転倒混和することによって溶解細胞。 </li><li>溶解中に、洗浄の間に2分間2700×gで40μlのGFP-トラップビーズの2つのロットの新鮮な溶解緩衝液で3回、沈降ビーズを洗います。 </li><li> 10分間21,000×gでの遠心分離によって溶解物を明確にします。 </li><li>転送は、4℃で転倒混和し、洗浄し、GFP-トラップビーズを分離し、GFP融合タンパク質は2時間結合させるための競合タンパク質のそれぞれの溶解液を明らかにしました。氷上でGFP-TrioD1細胞からの溶解物を保管してください。 </li><li> 50mMトリス-塩酸（pHは7.8）、50mMのNaClで二回ロードされたGFP-トラップビーズを洗浄し、0.7％（W / v）のノニデットP-40及び倍の50mMのTris-HCl（pHは7.6）で、20mMのMgCl 2を、洗浄の間に2分間2700×gでビーズを沈降。 </li><li> 40μlの0.2 Mグリシン（pHは2.5）を追加し、30秒間上下にピペッティングにより、GFP融合タンパク質を溶出させます。 4μlの1 Mトリス塩酸（pH値10.4）を含む新しいマイクロチューブに60秒と転送液の21,000×gですぐに土砂ビーズ。精製タンパク質への損傷を制限するために迅速にこれを行います。 </li><li>定量blottを使用して相対収率を確立するために、抗GFP抗体を用いたウェスタンブロットおよびプローブによる各精製タンパク質の1μLを分析製造業者のプロトコルに従ってシステムをる。別の方法として、ビシンコニン酸（BCA）アッセイによりタンパク質濃度を決定するが、タンパク質は同じ方法でアッセイと反応しないか、夾雑タンパク質が存在する場合、これはエラーを紹介します。 </li><li> 50mMトリス-塩酸（pHは7.6）、20mMのMgCl 2を添加することによってタンパク質のモル濃度を均一。 </li></ol> GTPアーゼの4ヌクレオチドの読み込み<ol><li>ステップ1で調製したGST-Rac1の磁気ビーズの1つのアリコートを解凍します。 </li><li> GST-Rac1のビーズの90μLを取り、20mMのトリス-HCl（pHは7.6）で3回洗浄し、各ステップでビーズを沈殿させ、磁性粒子ソーターを使用して、25mMの塩化ナトリウム、0.1mMのDTT、4mMのEDTA、。 </li><li>吸引除去ビーズからのバッファおよび25mMのNaCl、0.1mMのDTT、4 mMのEDTA、100μlの20 mMトリス - 塩酸（pHは7.6）を追加します。 </li><li> GDP、GTP又は全くヌクレオチド負荷が競合実験のために必要とされているかどうかによると、12μlの100 mMのGDP、12μlの10 mMのGUを追加anosine 5 &#39; - [γ-チオ]三リン酸（GTPγS）または60μlのGST-Rac1のビーズに無ヌクレオチド。 </li><li>ヌクレオチドローディングコントロールの場合、3 10-μlのアリコートに、残りのビーズを分割し、各チューブに2μlの100 mMのGDP、2μlの10 mMのGTPγSまたは全くヌクレオチドを​​追加します。 </li><li>ビーズを攪拌しながら30℃で30分間混合しインキュベートします。 </li><li>制御ミックス（ステップ4.5）のそれぞれに実験ミックス（ステップ4.4）に3μL、0.5μlの1 MのMgCl 2を添加することにより塩基結合するRac1を安定させます。 </li></ol> 5.競争が結合します。 <ol><li> 6マイクロチューブを設定し、それぞれ含みます： 200μlの50mMトリス-塩酸（pHは7.6）、20mMのMgCl 2を （ステップ4.7）10μlの実験ヌクレオチドロードRac1のビーズ 5μlのRac1の結合タンパク質A（定数結合タンパク質） </li><li>各チューブに、0、1、2.5、5、10または20μlのRac1の結合タンパク質B（可変結合タンパク質）を追加します。これらのボリュームは前提としていpproximately同等の株式定数と変数の結合タンパク質の濃度と調整する必要があります。 <ol><li>そこに2つのタンパク質の結合親和性に大きな差があり、これは実験の繰り返しを通じて、経験的に決定する必要がある場合は結合タンパク質AとBのボリュームを調整します。 50mMトリス-塩酸（pHは7.6）の添加によって235μlに結合混合物の全量を、20mMのMgCl 2を。 </li></ol></li><li>含むマイクロチューブを設定します。 200μlの50mMトリス-塩酸（pHは7.6）、20mMのMgCl 2を （ステップ4.7）10μlの実験ヌクレオチドロードRac1のビーズ 10μlのRac1の結合タンパク質A（定数結合タンパク質） </li><li> GDP、GTPγSなしヌクレオチド対照チューブを設定します。 200μlの50mMトリス-塩酸（pHは7.6）、20mMのMgCl 2を 10μlの制御Rac1のビーズは、GDP、GTPγSまたは無ヌクレオチドでステップ4.5でロードされ、ステップ4.7で安定しました。 180μlのHステップ3.6のように調製しEK293T GFP-TrioD1溶解物を、 4μlの1 MのMgCl 2 </li><li> 4℃での反転により混合、2時間混合物をインキュベートします。 </li><li> 50mMトリス-塩酸（pHは7.6）でビーズを3回洗浄し、20mMのMgCl 2を。 </li><li>溶出サンプルバッファーを減らす20μlのタンパク質を結合した​​（50mMのトリス-HCl（pHが7）、5％SDS、20％グリセロール、0.02 mg / mlのブロモフェノールブルー、5％βメルカプトエタノール）。 </li></ol>競争の6分析<ol><li>ドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳動（SDS-PAGE）およびウェスタンブロットによって、結合したタンパク質（ステップ5.6）の10μLを解決します。 </li><li>ブロッキング緩衝液中で1/1000に希釈した抗GFP抗体で4°CO / Nで膜をインキュベートすると、タグ付けされたGTPアーゼ結合タンパク質の両方を検出するために、PBS中0.1％のTween-20を1倍に希釈しました。 </li><li> PBS、0.1％のTween-20で10分間、膜を3回洗浄します。 </li><li> DyLight 800結合抗ウサギ秒で室温で30分間、膜をインキュベートブロッキング緩衝液中で1 /万希釈次側抗体は、PBS中の0.1％のTween-20を1倍に希釈しました。 </li><li> PBS、0.1％のTween-20で10分間、膜を3回洗浄します。 </li><li>製造業者のプロトコルに従ってバンド強度を測定するためのソフトウェアを使用して、赤外線イメージングシステムを用いて膜をスキャンします。 </li><li>変数競争相手（タンパク質B）の体積に対する各タンパク質のバンド強度をプロットします。 </li><li>線は平衡が達成された競技者の割合を決定するために、一定の競争相手（プロテインA、5μL）の体積交差する点で可変競技者の容積を分割します。 </li><li>ステップ6.1から6.6に記載されているように、ヌクレオチドローディング状態の検証については、p21活性化キナーゼ1（PAK1）（エフェクター）およびGFP-TrioD1（GEF）のための膜を探査。 </li></ol>

<ol>
	<li>Burridge, K., Rho Wennerberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=and+Rac+take+center+stage.">and Rac take center stage.</a> Cell. 116 (2), 167-179 (2004).</li><li>Raftopoulou, M., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+Rho+GTPases+lead+the+way.">Cell migration: Rho GTPases lead the way.</a> Dev Biol. 265 (1), 23-32 (2004).</li><li>Rossman, K. L., Der, C. J., Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEF+means+go:+turning+on+RHO+GTPases+with+guanine+nucleotide-exchange+factors.">GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.</a> Nat Rev Mol Cell Biol. 6 (2), 167-180 (2005).</li><li>Worthylake, D. K., Rossman, K. L., Crystal Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+Rac1+in+complex+with+the+guanine+nucleotide+exchange+region+of+Tiam1.">structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.</a> Nature. 408 (6813), 682-688 (2000).</li><li>Scheffzek, K., Ahmadian, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPase+activating+proteins:+structural+and+functional+insights+18+years+after+discovery.">GTPase activating proteins: structural and functional insights 18 years after discovery.</a> Cell Mol Life Sci. 62 (24), 3014-3038 (2005).</li><li>Del Pozo, ., A, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+regulate+GTP-Rac+localized+effector+interactions+through+dissociation+of+Rho-GDI.">Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI.</a> Nat Cell Biol. 4 (3), 232-239 (2002).</li><li>Williamson, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin-1C+and+RCC2+guide+mesenchymal+migration+by+trafficking+Rac1+and+controlling+GEF+exposure.">Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure.</a> J Cell Sci. 127 (Pt 19), 4292-4307 (2014).</li><li>Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+to+the+extracellular+matrix+regulates+the+coupling+of+the+small+GTPase+Rac+to+its+effector+PAK.">Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK.</a> Embo J. 19 (9), 2008-2014 (2000).</li><li>Van Rijssel, J., Hoogenboezem, M., Wester, L., Hordijk, P. L., Van Buul, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-terminal+DH-PH+domain+of+Trio+induces+cell+spreading+and+migration+by+regulating+lamellipodia+dynamics+in+a+Rac1-dependent+fashion.">The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion.</a> PLoS. 7 (1), e29912 (2012).</li><li>Self, A. J., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intrinsic+nucleotide+exchange+and+GTP+hydrolysis+rates.">Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates.</a> Methods Enzymol. 256, 67-76 (1995).</li></ol>

The authors have nothing to disclose.

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.

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&#8217;-triphosphate (GTP)-bound GTPases activate downstream effectors, whereas the Guanosine 5&#8217;-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.

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			<td>Bugbuster</td>
			<td>Novagen</td>
			<td>70584-3</td>
			<td></td>
		</tr>
		<tr>
			<td>COMPLETE protease inhibitor</td>
			<td>Roche</td>
			<td>05 056 489 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione magnetic beads</td>
			<td>Pierce</td>
			<td align="right">88821</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched, average Mw ~25,000</td>
			<td>Sigma Aldrich</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>OPIMEM</td>
			<td>Life Technologies</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10270-1-6</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Trap_A</td>
			<td>Chromotec</td>
			<td>gta-20</td>
			<td></td>
		</tr>
		<tr>
			<td>GDP</td>
			<td>Sigma Aldrich</td>
			<td>G7127</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>GTP&#947;S</td>
			<td>Sigma Aldrich</td>
			<td>G8634</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>Sigma Aldrich</td>
			<td>B6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Living Colors</td>
			<td align="right">632592</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>DyLight 800 conjugated goat anti-rabbit secondary antibody</td>
			<td>Fisher Scientific</td>
			<td align="right">10733944</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAK1 antibody</td>
			<td>Cell Signaling</td>
			<td>2602S</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>Odyssey SA Infrared Imaging System</td>
			<td>Li-cor</td>
			<td>9260-11PC</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparing the affinity of gtpase-binding proteins using competition assays

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.

This protocol is designed to calculate the relative affinities of binding partners for Rac1, without the need to know the precise concentration of the competitors (Figure 1). Determination of protein concentration introduces errors and when considering competition between molecules in a signaling pathway is not needed. However, it is important to know that the two competitors have the same molar concentration in the stock solutions to allow simple ratios to be calculated when adding different volumes to the assay. 40 &#181;l of GFP-Trap beads have a binding capacity of ~300 pmol so a confluent 75 cm2 flasks of highly expressing cells will saturate the beads, with the result that the preparations of the two different binding proteins will be similar before adjustment (Figure 2A). If one of the proteins expresses poorly, this problem can be overcome by purifying that protein from more than one flask of cells.
The binding of most GTPase effectors and regulators depends on the nucleotide-loading of the bait GTPase, so it is important to test whether loading has been successful. Loading can be verified by precipitating known binding proteins from cell lysates. Effector proteins, such as PAK1 bind to GTP-Rac1 and can be easily precipitated from lysates and detected by Western blotting 8 (Figure 2B). GEFs bind preferentially to nucleotide-free GTPase to stabilize the transition state. As GEFs are of low abundance, usually inactive and frequently blot poorly, it is better to overexpress a GEF or GEF fragment to test nucleotide-free GTPase. We frequently use the first Dbl homology of Trio, expressed as a GFP fusion (GFP-TrioD1 9) (Figure 2B) but any GEF would work. Proteins that bind to the GDP-loaded GTPase are rarer. We recently reported RCC2 as one such protein 7, or GDP-loading can be validated simply as binding to neither GEF nor effector.
The output from the experiment will be a Western blot depicting the two GFP-tagged binding partners bound to the GTPase. By using a single antibody to detect both proteins, the concentrations at which similar amounts of both competitors bind can be determined and therefore the relative affinities inferred. In this example competition between the propeller domain of the Rac1-trafficking protein, coronin-1C (Rac1-binding protein A), and the Rac1-sequestering protein, RCC2 (Rac1-binding protein B), is demonstrated (Figure 3A). By using a constant volume of coronin-1C propeller (5 &#181;l), and adding increasing volumes of RCC2, we can see from the GFP blot that equilibrium is reached at 1.25-2.5 &#181;l of RCC2 (asterisk), demonstrating that RCC2 has a stronger affinity for Rac1 than coronin-1C. By measuring the intensity of bands using quantitative Western blotting, and plotting average values for each competitor, the equilibrium point can be calculated accurately by identifying the volumes at which the curves intersect (Figure 3B).
One of the possible obstacles to a successful competition assay is if the binding partners bind to one another as well as binding to Rac1. In Figure 3A+B we demonstrate competition between RCC2 and the propeller domain of coronin-1C, rather than full-length coronin-1C. The reason for using the truncated coronin is that coronin-1C also binds RCC2 through the tail domain. When full-length coronin-1C is titrated against RCC2, binding of both proteins is detected, due to ternary complex formation, rather than competition (Figure 3C). If competition is occurring, binding of one protein will increase while the other decreases, and total bound GFP-fusion will remain constant. In cases where a ternary complex forms it is necessary to truncate one of the GTPase-binding protein so that the competitors no longer interact.
<img alt="Figure 1" src="/files/ftp_upload/53254/53254fig1.jpg" /> 
Figure 1.&#160;Workflow. Schematic representation of the workflow for determining the affinity of GTPase-binding proteins using competition assays. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53254/53254fig2.jpg" /> 
Figure 2. Validation of purified proteins. (A) Purified GFP-tagged Rac1 binding proteins analyzed by Western blot, probing with anti-GFP to determine the relative yield of the two proteins. This type of equalization during the experiment allows the concentration of the two proteins to be adjusted so they match in the binding experiment. (B) GDP, GTP&#947;S and no nucleotide-loaded GST-Rac1 was incubated with lysate from HEK293T expressing GFP-TrioD1 and bound proteins detected by plotting for endogenous PAK1 or overexpressed GFP-TrioD1. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53254/53254fig3.jpg" /> 
Figure 3. Western blot analysis of relative protein binding. Example outputs from competition-binding assays. (A) GDP-loaded Rac1 was mixed with 5 &#181;l GFP-coronin-1C propeller domain and increasing volumes of GFP-RCC2 were titrated in. By Western blotting bound proteins for GFP, issues with differential detection of the two proteins are avoided and the GFP signal reports the molar ratio between the two fusion proteins. Asterisks indicate the competition ratios on either side of the equilibrium point. (B) Band intensities of bound GFP fusion proteins from three independent experiments were measured by quantitative Western blotting, using fluorophore-conjugated secondary antibodies and averages plotted to calculate the amount of RCC2 needed to reach equilibrium. (C) Example output from an experiment where Rac1-binding proteins bind to one another and form a ternary complex, rather than competing. GDP-loaded Rac1 was mixed with 5 &#181;l GFP-RCC2 and increasing volumes of GFP-coronin-1C full-length were titrated in. The increase in bound GFP-coronin-1C without loss of bound GFP-RCC2 indicates ternary complex formation. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Comparing the Affinity of GTPase-binding Proteins using Competition Assays at JoVE.com

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

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.

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