A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins

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Molecular Microbiology
Dec 2013



Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many processes in bacteria including biofilm formation, motility, and virulence (Hengge, 2009). Analysis of c-di-GMP binding properties of bacterial proteins is an important step to characterize c-di-GMP signaling pathways. C-di-GMP binds numerous proteins such as transcription factors, enzymes, and multimeric protein complexes (Hickman and Harwood, 2008, Ryjenkov et al., 2006, Weinhouse et al., 1997). The c-di-GMP binding assay described here is a relatively simple and cost effective method to characterize c-di-GMP binding to a protein using [32P]-labeled c-di-GMP. Radiolabeled c-di-GMP is readily synthesized with a purified GGDEF enzyme [such as WspR from Pseudomonas aeruginosa (P. aeruginosa)] and [32P]-GTP (Srivastava et al., 2013). After incubation of the labeled c-di-GMP with the protein of interest in solution, the resulting mixture is filtered through a nitrocellulose protein binding membrane. The amount of labeled c-di-GMP that is retained on the membrane indicates the interaction between the signal and protein. The specificity of c-di-GMP binding can be tested by competing with unlabeled c-di-GMP or other nucleotides such as GTP in the reaction. By examining binding of a fixed protein concentration to increasing concentrations of c-di-GMP, this method is able to determine the dissociation constant of c-di-GMP-protein interaction.

Keywords: Cyclic-di-GMP (环状二GMP), Ligand binding (配体结合), Affinity (密切关系), Kd (KD), Protein c-di-GMP interaction (蛋白质相互作用的调控)

Materials and Reagents

  1. Purified proteins to be tested
    Note: Any kind of buffer can be used to prepare protein samples. We have tested this for sodium phosphate and Tris based buffers.
  2. [α-32P]-GTP (800 Ci/mmol, 10 mCi/ml, 250 µCi) (PerkinElmer, catalog number: BLU006X250UC )
  3. c-di-GMP unlabeled (Axxora)
  4. GTP (Sigma-Aldrich, catalog number: 51120 )
  5. Purified diguanylate cyclase [WspR (R242A mutant), P. aeruginosa (Sambanthamoorthy et al., 2012)]
  6. Antarctic phosphatase (New England Biolabs, catalog number: M0289S )
  7. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  8. Bradford reagent for protein concentration estimation (Bio-Rad Laboratories, catalog number: 500-0205 )
  9. Safety-SolveTM complete counting cocktail (RPI, catalog number: 111177 )
  10. Protein purification buffer (see Recipes)
  11. Binding buffer (see Recipes)


  1. Nitrocellulose paper (0.2 µm) (Whatman, Optitran®, catalog number: BA-S 83 )
  2. Whatman filter paper (Whatman, catalog number: 3MM )
  3. Radioactive working area containing a centrifuge and heat block
  4. Scintillation vials (20 ml, with Poly screw caps, case of 500) (PerkinElmer, Econo Glass Vial, catalog number: 6000097)
  5. Scintillation counter (Beckman Coulter, model: LS 6500 )
  6. Biorad slot blot (Hybri Dot Manifold) (Bristol Robotics Laboratory, catalog number: 1050MM )
  7. 1.5 ml eppendorf tubes
  8. Filter tips
  9. Forceps and scissors


  1. GraphPad Prism 5.0


  1. Proteins to be tested are purified using the manufacturer’s instructions. We have used this assay to test binding to both 6x HIS tagged proteins (cloned and expressed from pET28b vectors) and untagged protein (cloned and expressed from pTXB1). Purified proteins are quantified using a Bradford assay (Bio-Rad Laboratories).
  2. A positive control protein that is known to bind to c-di-GMP such as YcgR from Escherichia coli should be included in the experiment. The purification of the positive control is performed similar to the test protein. BSA can be used as a negative control.
  3. Radiolabeled c-di-GMP is synthesized using a purified diguanylate cyclase enzyme with [α-32P]-GTP as a substrate. We have successfully used WspR (R242A mutant), a GGDEF protein from Pseudomonas aeruginosa for c-di-GMP synthesis (Sambanthamoorthy et al., 2012). The activity of the enzyme is tested as described (Hunter et al., 2014).
  4. Before starting the experiment, all buffers should be prepared and adjusted to the appropriate pH as described below.
  5. Synthesis of labeled c-di-GMP
    1. Thaw the [α-32P]-GTP.
    2. The reaction for c-di-GMP synthesis is set up as shown below:
      Volume (µl)
      NaCl (1 M)
      Tris (1 M, pH 7.6)
      MgCl2 (1 M)
      [α-32P]-GTP or unlabeled GTP (12.5 µM)
      ~100 μM
      *The activity of the enzyme is previously determined to assess the amount to be added.
      The total volume of the reaction is 50 µl.
    3. A reaction using unlabeled c-di-GMP is performed side by side to the reaction using labeled c-di-GMP for subsequent quantification of reaction efficiency.
    4. The reaction is incubated at room temperature overnight.
    5. 1 µl of Antarctic phosphatase (NEB) is added to the reaction to hydrolyze the leftover [α-32P]-GTP.
    6. The reaction is boiled for 10 min at 100 °C and precipitated protein is removing by centrifugation at 16,000 x g for 10 min at room temperature followed by removal of the supernatant.
    7. The reaction is transferred to another eppendorf tube and stored at -20 °C until further use.
  6. The concentration of c-di-GMP in the unlabeled control reaction is measured using ultra high-pressure liquid chromatography (UPLC)–tandem mass spectrometry (MS-MS) as previously described (Massie et al., 2012). The labeled reaction is assumed to be equivalent. Typically, the in vitro diguanylate cyclase reaction generates near 100% maximum yield based on the starting concentration of c-di-GMP.
  7. For binding reactions, 100 to 500 nM protein is incubated with varying amounts of [32P]-c-di-GMP (0.125 µM to 1.5 µM) in a 20 µl volume as shown below for 30 min at room temperature. The range of labeled c-di-GMP used is dependent on the particular binding parameters for the test protein. Each sample is tested in duplicate. Equivalent amounts of YcgR and BSA are included as positive and negative controls, respectively.
    Amount/Volume (µl)
    100-500 nM
    Binding buffer
    [α-32P] c-di-GMP
    0.125 µM to 1.5 µM
    Up to 20 µl

  8. As the binding reactions are incubating, the slot blot apparatus is prepared. A nitrocellulose membrane and whatman filter paper are cut to size of the dot blot assembly. The membrane and filter paper are immersed briefly in binding buffer to wet them. The dot blot apparatus is connected to vacuum and the membrane and filter paper are placed on the pore side with the vacuum turned on. The assembly is then completed by securely screwing in the top layer of the apparatus. This assures the correct vacuum sealing of the apparatus.

    Figure 1. Hybri dot manifold assembly

  9. Preparations are then loaded onto a nitrocellulose membrane through a vacuum dot blot. The samples loaded on the dot blot should be well separated (one well on each side of sample well should be empty). The orientation of the blot should be marked before loading. This prevents contamination of counts during the processing step. After 5 min of sample loading, the sample wells are washed with 3 ml binding buffer (adding 200 µl of binding buffer at a time, 15 subsequent washes) in order to wash away unbound [32P]-c-di-GMP. The vacuum is kept on for 5 min after washing to dry the assembly out.
  10. The membrane is then removed from assembly and dried for 15 min on a dry filter paper, the spots containing sample are cut into square pieces and added to 5 ml scintillation fluid in a scintillation vial.
  11. The bound [32P]-c-di-GMP of individual wells is quantified by liquid scintillation counting (cpm/min).
  12. For competition experiments with unlabeled c-di-GMP and GTP, following incubation of the protein with [32P]-c-di-GMP for 15 min, 3 µM unlabeled nucleotides (unlabeled c-di-GMP and GTP) are added and the reaction is incubated for another 15 min and processed similarly.
  13. The data from the binding experiments is analyzed with GraphPad Prism 5.0 using non-linear regression analysis [a representative experiment is shown in Srivastava et al. (2013)].

Representative data

A representative experiment demonstrating c-di-GMP binding to V. cholerae transcription factor FlrA is shown in Srivastava et al. (2013).


  1. We have tested this protocol for transcription factors, which are mostly cytoplasmic proteins. But, in principle, this protocol should work for any bacterial proteins.
  2. To ensure that the Slot Blot assembly is securely tightened, use a dilute colored dye solution (such as Bromophenol Blue) to run through a few interspersed wells. The color should remain within the well and not spread out if the slot blot is sealed well and no bleed through is occurring between wells.


  1. Protein purification buffer
    As per the manufacturer’s directions
  2. Binding buffer
    10 mM MgCl2
    20 mM Tris (pH 7.8)
    50 mM NaCl


This protocol was modified and adapted from a binding assay described previously (Hickman and Harwood, 2008).


  1. Hengge, R. (2009). Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 7(4): 263-273.
  2. Hickman, J. W. and Harwood, C. S. (2008). Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 69(2): 376-389.
  3. Hunter, J. L., Severin, G. B., Koestler, B. J. and Waters, C. M. (2014). The Vibrio cholerae diguanylate cyclase VCA0965 has an AGDEF active site and synthesizes cyclic di-GMP. BMC Microbiol 14: 22.
  4. Massie, J. P., Reynolds, E. L., Koestler, B. J., Cong, J. P., Agostoni, M. and Waters, C. M. (2012). Quantification of high-specificity cyclic diguanylate signaling. Proc Natl Acad Sci U S A 109(31): 12746-12751.
  5. Ryjenkov, D. A., Simm, R., Römling, U. and Gomelsky, M. (2006). The PilZ domain is a receptor for the second messenger c-di-GMP THE PilZ DOMAIN PROTEIN YcgR CONTROLS MOTILITY IN ENTEROBACTERIA. J Biol Chem 281(41): 30310-30314.
  6. Sambanthamoorthy, K., Sloup, R. E., Parashar, V., Smith, J. M., Kim, E. E., Semmelhack, M. F., Neiditch, M. B. and Waters, C. M. (2012). Identification of small molecules that antagonize diguanylate cyclase enzymes to inhibit biofilm formation. Antimicrob Agents Ch 56(10): 5202-5211.
  7. Srivastava, D., Hsieh, M. L., Khataokar, A., Neiditch, M. B. and Waters, C. M. (2013). Cyclic di-GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production. Mol Microbiol 90(6): 1262-1276.
  8. Weinhouse, H., Sapir, S., Amikam, D., Shilo, Y., Volman, G., Ohana, P. and Benziman, M. (1997). c-di-GMP-binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum. FEBS Lett 416(2): 207-211.


循环di-GMP(c-di-GMP)是一种普遍存在的第二信使,其调节细菌中的许多过程,包括生物膜形成,运动性和毒力(Hengge,2009)。细菌蛋白的c-di-GMP结合性质的分析是表征c-di-GMP信号传导途径的重要步骤。 C-di-GMP结合许多蛋白质,例如转录因子,酶和多聚体蛋白复合物(Hickman和Harwood,2008,Ryjenkov等人,2006,Weinhouse等人>,1997)。本文所述的c-di-GMP结合测定法是使用[32 P] - 标记的c-di-GMP表征c-di-GMP与蛋白质结合的相对简单且成本有效的方法。放射性标记的c-di-GMP易于用纯化的GGDEF酶[例如来自绿脓假单胞菌(铜绿假单胞菌)(绿脓杆菌)]的WspR和[<32> > P] -GTP(Srivastava等人,2013)。在标记的c-di-GMP与溶液中目标蛋白温育后,将所得混合物通过硝酸纤维素蛋白结合膜过滤。保留在膜上的标记的c-di-GMP的量指示信号和蛋白质之间的相互作用。可以通过与未标记的c-di-GMP或其他核苷酸(例如反应中的GTP)竞争来测试c-di-GMP结合的特异性。通过检测固定蛋白浓度与增加浓度的c-di-GMP的结合,该方法能够确定c-di-GMP-蛋白相互作用的解离常数。

关键字:环状二GMP, 配体结合, 密切关系, KD, 蛋白质相互作用的调控


  1. 要测试的纯化蛋白质
    注意:任何类型的缓冲液可用于制备蛋白质样品。 我们已经测试了磷酸钠和Tris缓冲液。
  2. (PerkinElmer,目录号:BLU006X250UC)[γ-32 P] -GTP(800Ci/mmol,10mCi/ml,250μCi)
  3. c-di-GMP unlabeled(Axxora)
  4. GTP(Sigma-Aldrich,目录号:51120)
  5. 纯化的二鸟苷酸环化酶[WspR(R242A突变体),P。 铜绿(Sambanthamoorthy ,2012)]
  6. 南极磷酸酶(New England Biolabs,目录号:M0289S)
  7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
  8. 用于蛋白质浓度估计的Bradford试剂(Bio-Rad Laboratories,目录号:500-0205)
  9. Safety-Solve TM 完全计数鸡尾酒(RPI,目录号:111177)
  10. 蛋白质纯化缓冲液(参见配方)
  11. 绑定缓冲区(参见配方)


  1. 硝化纤维素纸(0.2μm)(Whatman,Optitran ,目录号:BA-S 83)
  2. Whatman滤纸(Whatman,目录号:3MM)
  3. 包含离心机和加热块的放射性工作区域
  4. 闪烁瓶(20ml,具有Poly螺旋盖,500的情况)(PerkinElmer,Econo Glass Vial,目录号:6000097)
  5. 闪烁计数器(Beckman Coulter,型号:LS 6500)
  6. Biorad槽印迹(Hybri Dot Manifold)(Bristol Robotics Laboratory,目录号:1050MM)
  7. 1.5 ml eppendorf管
  8. 过滤提示
  9. 镊子和剪刀


  1. GraphPad Prism 5.0


  1. 使用制造商的说明书纯化待测试的蛋白质。我们使用该测定来测试对6x HIS标记的蛋白(从pET28b载体克隆和表达)和未标记的蛋白(从pTXB1克隆和表达)的结合。使用Bradford测定法(Bio-Rad Laboratories)对纯化的蛋白质进行定量。
  2. 应该包括已知结合c-di-GMP的阳性对照蛋白,例如来自大肠杆菌的YcgR。阳性对照的纯化类似于测试蛋白进行。 BSA可用作阴性对照。
  3. 放射性标记的c-di-GMP使用具有[α-32 P] -GTP作为底物的纯化的二鸟苷酸环化酶合成。我们已经成功地使用来自绿脓假单胞菌的GGDEF蛋白的WspR(R242A突变体)用于c-di-GMP合成(Sambanthamoorthy等人,2012)。如(Hunter et al。,2014)所述测试酶的活性。
  4. 在开始实验之前,应制备所有缓冲液,并如下所述调节至适当的pH。
  5. 标记的c-di-GMP的合成
    1. 解冻[α- 32 P] -GTP。
    2. c-di-GMP合成的反应如下所示:
      Tris(1M,pH 7.6) 1.2
      (Mg)2/MgCl 2(1M) 0.25
      [α-32 P] -GTP或未标记的GTP(12.5μM)
      WspR *
      *预先确定酶的活性以评估待添加的量 反应的总体积为50μl
    3. 使用未标记的c-di-GMP的反应并行进行   反应使用标记的c-di-GMP进行随后的定量 反应效率。
    4. 将反应在室温下温育过夜。
    5. 向反应中加入1μl的南极磷酸酶(NEB)以水解剩余的[α-32 P] -GTP。
    6. 将反应在100℃下煮沸10分钟并沉淀蛋白质  通过在室温下以16,000×g离心10分钟除去 温度,然后除去上清液
    7. 将反应物转移到另一个eppendorf管中,并在-20℃下储存,直到进一步使用。
  6. 使用如先前所述的超高压液相色谱(UPLC)串联质谱(MS-MS)测量未标记的对照反应中c-di-GMP的浓度(Massie等人, 2012)。假定标记的反应是等效的。通常,体外二瓜氨酸环化酶反应基于c-di-GMP的起始浓度产生接近100%的最大产量。
  7. 对于结合反应,将100-500nM蛋白与如下所示的20μl体积中的不同量的[32 P] -c-二-GMP(0.125μM至1.5μM)温育30分钟。使用的标记的c-di-GMP的范围取决于测试蛋白的特定结合参数。每个样品一式两份测试。包括等量的YcgR和BSA作为阳性和阴性对照。
    100-500 nM
    [α- 32 P] c-di-GMP


  8. 当结合反应孵育时,制备狭缝印迹装置。将硝酸纤维素膜和Whatman滤纸切割成斑点印迹组件的尺寸。将膜和滤纸短暂地浸入结合缓冲液中以润湿它们。将斑点印迹装置连接到真空,并将膜和滤纸放置在孔侧并打开真空。然后通过牢固地拧入装置的顶层来完成组件。这保证了设备的正确的真空密封

    图1. Hybri点歧管组件

  9. 然后通过真空斑点印迹将制剂装载到硝酸纤维素膜上。装载在斑点印迹上的样品应该很好地分离(样品孔每侧上的一个孔应该是空的)。在装载前应标记印迹的方向。这防止在处理步骤期间污染计数。在样品加载5分钟后,用3ml结合缓冲液(每次加入200μl结合缓冲液, 15次洗涤),以洗去未结合的[32 P] -c-二-GMP。在洗涤后保持真空5分钟以干燥组件。
  10. 然后将膜从组件中取出并在干燥的滤纸上干燥15分钟,将含有样品的斑点切成方块,并加入到闪烁瓶中的5ml闪烁液中。
  11. 通过液体闪烁计数(cpm/min)对各孔的结合的[32 P] -c-di-GMP进行定量。
  12. 对于使用未标记的c-di-GMP和GTP的竞争实验,在蛋白质与[32 P] -c-二-GMP孵育15分钟后,将3μM未标记的核苷酸(未标记的c- GMP和GTP),并将反应物再温育15分钟并类似地处理
  13. 使用非线性回归分析,使用GraphPad Prism 5.0分析来自结合实验的数据[代表性实验显示在Srivastava等人(2013)]。


代表性实验证明c-di-GMP结合于V。 霍乱弧菌转录因子FlrA在Srivastava等人(2013)中显示。


  1. 我们已经测试这种协议的转录因子,大多是细胞质蛋白。 但是,在原则上,这个协议应该适用于任何细菌蛋白质
  2. 为了确保槽缝印刷组件安全紧固,使用稀释的染色溶液(如溴酚蓝)穿过几个散置的孔。 如果狭缝印迹很好地密封并且在孔之间不发生渗出,则颜色应当保持在孔内并且不扩散。


  1. 蛋白纯化缓冲液
  2. 绑定缓冲区
    10mM MgCl 2/
    20mM Tris(pH7.8)
    50mM NaCl




  1. Hengge,R。(2009)。 细菌中c-di-GMP信号传导的原理。 Nat Rev Microbiol 7(4):263-273。
  2. Hickman,J.W。和Harwood,C.S。(2008)。 从绿脓假单胞菌中鉴定FleQ作为c-di-GMP- response transcription factor。 Mol Microbiol 69(2):376-389
  3. Hunter,J.L.,Severin,G.B.,Koestler,B.J.and Waters,C.M。(2014)。 霍乱弧菌二鸟苷酸环化酶VCA0965具有AGDEF活性位点并合成环状di-GMP。 BMC Microbiol 14:22.
  4. Massie,J.P.,Reynolds,E.L.,Koestler,B.J.,Cong,J.P.,Agostoni,M.and Waters,C.M。(2012)。 高特异性循环二乙烯化物信号的定量。美国国家科学院 109(31):12746-12751。
  5. Ryjenkov,D.A.,Simm,R.,Römling,U。和Gomelsky,M。(2006)。 PilZ域是第二个信使的受体c-di-GMP THE PilZ DOMAIN 蛋白YcgR控制肠球菌的运动性。 J Biol Chem 281(41):30310-30314。
  6. Sambanthamoorthy,K.,Sloup,R.E.,Parashar,V.,Smith,J.M.,Kim,E.E.,Semmelhack,M.F.,Neiditch,M.B.and Waters,C.M。(2012)。 拮抗双胍酸环化酶抑制生物膜形成的小分子的鉴定。 em> Antimicrob Agents Ch 56(10):5202-5211。
  7. Srivastava,D.,Hsieh,M.L.,Khataokar,A.,Neiditch,M.B.and Waters,C.M。(2013)。 循环di-GMP通过抑制转录诱导而抑制霍乱弧菌的运动性,诱导细胞外多糖生产。 Mol Microbiol 90(6):1262-1276。
  8. Weinhouse,H.,Sapir,S.,Amikam,D.,Shilo,Y.,Volman,G.,Ohana,P.and Benziman,M。(1997)。 c-di-GMP结合蛋白,调节木醋杆菌中纤维素合成的新因子。 FEBS Lett 416(2):207-211。
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引用:Srivastava, D. and Waters, C. M. (2015). A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins. Bio-protocol 5(3): e1394. DOI: 10.21769/BioProtoc.1394.