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Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay
荧光热变分析测定假激酶配体的相互作用   

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Abstract

This protocol describes a robust technique for the measurement of pseudokinase-ligand interaction by a fluorescence-based Thermal Shift Assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulators of signal transduction and may therefore represent a novel class of drug targets. Unlike kinases, the catalytic efficiency of pseudokinases is rather poor or non existent, making it difficult to dissect the function of their nucleotide binding sites. Thermal denaturation-based methods have proven to be a powerful method for determining ligand binding capacity to purified pseudokinases and can inform on the potential drugability of the nucleotide binding site. This assay takes advantage of a change in flurorescence arising when a flurorescence dye, in this instance SYPRO® Orange, binds to hydrohobic patches that become exposed when a protein undergoes thermal denaturation. Ligand binding to a protein is known to increase its thermal stability which is reflected by a shift observed in the thermal denaturation curve between the unliganded protein and the liganded protein. This generalized protocol can also be tailored to other protein families. In addition, thermal denaturation-based methods can be used to identify optimal buffer conditions that may increase protein stability.

Materials and Reagents

  1. Purified protein (stock solution preferably at a concentration above 20 μM) (Murphy et al., 2013)
  2. 1 M Dithiothreitol (DTT) stock (Astral Scientific, catalog number: C-1029 )
  3. DMSO (high grade) (Sigma-Aldrich, catalog number: D-1435 )
  4. MilliQ water
  5. Nucleotide solutions (10 mM stock prepared in 20 mM Tris) (pH 8)
    1. ATP (Sigma-Aldrich, catalog number: A2383 )
    2. ADP (Sigma-Aldrich, catalog number: A2754 )
    3. AMP-PNP (Sigma-Aldrich, catalog number: A2647 )
    4. GTP (Sigma-Aldrich, catalog number: G8877 )
  6. Divalent cations salt solutions (50 mM stock in MilliQ water)
    1. MnCl2 (Sigma-Aldrich, catalog number: 203734 )
    2. MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  7. Kinase inhibitor solutions (2 mM stock in 100% DMSO) (such as the pan-kinase inhibitors, Staurosporine, Sigma-Aldrich, catalog number: S5921 )
  8. Thermal Shift Assay buffer (see Recipes)
  9. SYPRO® Orange (Sigma-Aldrich, catalog number: S5692 ) (see Recipes)

Equipment

  1. RT-PCR tubes GST-RG01 (Gene Targets Solutions)
  2. 1.5 ml microfuge tube
  3. Qiagen/Corbett Rotor-Gene® 3000 RT-PCR machine (QIAGEN) (Murphy et al., 2013)

Software

  1. Microsoft Excel or Prism

Procedure

  1. Thermal Shift Assay (TSA) test run for the determination of optimal amount of protein
    1. Prepare a dilution series of your protein sample in buffer ranging from 1 to 10 μM final concentration in a total volume of 25 μl. Add 1 μl of 1x SYPRO® Orange to each tube.
    2. Perform a thermal cycler run using the parameters as described in step 3. Analyze the melt-curve (see step 4) and determine the optimal amount of protein that gives at least 30 fluorescence unit. Reduce amount of protein if the fluorescence signal is saturated.
      Notes: Typical final protein concentration is around 5 μM. The Rotor-Gene® 3000 is equiped  with a gain optimization function that can be adjusted for optimal signal (refer to manufacturer’s manual).
  2. Assay
    1. Start by defining the number of conditions that you need to test, including appropriate controls such as protein sample in buffer only if testing nucleotide binding, or in presence of DMSO if testing compounds. This will determine the total volume of master mix of protein needed to conduct the entire experiment. Each reaction is conducted in a total volume of 25 μl.
    2. Prepare on ice just prior use a master mix containing your protein in the Thermal Shift Assay buffer using the concentration of protein that gave the peak fluorescence (without signal saturation) in step 1.
    3. Dispense 23 μl of protein/buffer, add 1 μl of ligand (10 to 200 μM) or 1 μl of DMSO/buffer for the control experiment and 1 μl of diluted SYPRO® Orange (1:100). Prepare tubes in duplicate. If the protein to test is highly unstable at room temperature, it is recommended to prepare the tubes on ice. Nucleotide solutions are typically used at a final concentration of 200 μM final, divalent cations at 1 mM final and kinase inhibitors at 40 μM final.
  3. Thermal cycler program
    1. Fluorescence-based Thermal Shift Assay can be performed using instruments that combine both sample temperature control and dye fluorescence detection. In this instance, we used the Qiagen/Corbett Rotor-Gene® 3000 RT-PCR machine.
    2. Start at 25 °C, hold at 25 °C for 2 min, increase temperature of 1 °C/min for 65 cycles (25 °C to 90 °C) reading fluorescence intensity every °C. Return to 25 °C. Excitation is at 470 nm (green chanel) and emission is at 555 nm (yellow chanel).
  4. Data analysis
    1. Save data as fluorescence intensity vs temperature. Export file in a format suitable for import into Microsoft Excel or Prism for analyses.
    2. The highest fluorescence density is used as a cutoff for the data, therefore remove all data after the high fluroescence density peak. Fit fluorescence intensity curve to a Boltzmann sigmoidal curve using Prism.
    3. Obtain the melting temperature (Tm) of the protein in buffer/DMSO, which correspond to the midpoint for the protein unfolding curve. Similarly, obtain the Tm of the protein when ligand is added.
    4. Calculate ΔTm = Tm ligand - Tm buffer/DMSO. A positive ΔTm indicates that the ligand stabilizes the protein from denaturation, hence binds to the protein. A value ≥ to 2-3 °C is an indicator of ligand binding.
    5. Below is an example of a typical TSA experiment.
      Notes: If PRISM software is not available, non-linear regression analysis of experimental data can be conducted using a Microsoft Excel spreadsheet (Brown, 2001).


    Figure 1. Practical example of a TSA experiment. (Top) Thermal Shift Assay data were obtained for mouse MLKL in the presence and absence of nucleotides. The midpoint for the unfolding transition in the absence of ligand was observed to be 44.8 °C while the Tm shifted to 53.8 °C in presence of ATP, inducing a shift of 9 °C. (Bottom) Table summarizing the Tm and ΔTm values obtained for mMLKL in presence of ATP, GTP and AMPPNP.

Recipes

  1. Thermal Shift Assay buffer
    The composition of the buffer used to dilute the protein can be optimised according to each protein. The following recipe was used to study pseudokinases.
    20 mM Tris (pH 8)
    150 mM NaCl
    1 mM DTT
  2. SYPRO® Orange
    1 to 100 dilution in 100% high grade DMSO

Notes

  1. While the thermal denaturation of a single domain proteins mostly results in a single transition phase, multidomain proteins may exhibit distinct transition states. Nevertheless if a ligand binds tightly to one domain, it could result in a change in the thermal denaturation profile, providing a tractable method for assaying ligand binding. There are also cases in which a ligand may bind tightly but does not induce a change in the Tm.
  2. The protocol described has been adapted to the Qiagen/Corbett Rotor-Gene® 3000 RT-PCR machine. The amount of protein required per experiment and the SYPRO® Orange dilution may need to be adapted depending on the RT-PCR machine used.

Acknowledgments

We thank the Monash University Protein Production Unit for access to the Corbett RT-PCR instrument used for development of the thermal-shift assay. This work was supported by National Health and Medical Research Council (NHMRC) grants (1016647, 461221, 1016701, 637342, 1025594, 1046984) and fellowships to J.M.H., N.A.N. and J.S.; Australian Research Council fellowships to P.E.C., J.J.B. and J.M.M.; and additional support from the Australian Cancer Research Fund, Victorian State Government Operational Infrastructure Support, and NHMRC IRIISS grant (361646). This assay was developed over the course of completing the following studies: Murphy et al. (2013) and Murphy et al. (2014).

References

  1. Brown, A. M. (2001). A step-by-step guide to non-linear regression analysis of experimental data using a Microsoft Excel spreadsheet. Comput Methods Programs Biomed 65(3): 191-200.
  2. James, M. M., Isabelle, S. L., Joanne, M. H., Maria, C. T., Samuel, N. Y., Pooja, S., Guillaume, L., Warren, S. A., Jeffrey, J. B. and John, S. (2014). Insights into the evolution of divergent nucleotide-binding mechanisms among pseudokinases revealed by crystal structures of human and mouse MLKL. Biochem J 457(3): 369-377.
  3. Murphy, J. M., Czabotar, P. E., Hildebrand, J. M., Lucet, I. S., Zhang, J. G., Alvarez-Diaz, S., Lewis, R., Lalaoui, N., Metcalf, D., Webb, A. I., Young, S. N., Varghese, L. N., Tannahill, G. M., Hatchell, E. C., Majewski, I. J., Okamoto, T., Dobson, R. C., Hilton, D. J., Babon, J. J., Nicola, N. A., Strasser, A., Silke, J. and Alexander, W. S. (2013). The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39(3): 443-453.
  4. Murphy, J. M., Zhang, Q., Young, S. N., Reese, M. L., Bailey, F. P., Eyers, P. A., Ungureanu, D., Hammaren, H., Silvennoinen, O., Varghese, L. N., Chen, K., Tripaydonis, A., Jura, N., Fukuda, K., Qin, J., Nimchuk, Z., Mudgett, M. B., Elowe, S., Gee, C. L., Liu, L., Daly, R. J., Manning, G., Babon, J. J. and Lucet, I. S. (2014). A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. Biochem J 457(2): 323-334.

简介

该协议描述了通过基于荧光的热漂移测定(TSA)测量假激酶 - 配体相互作用的鲁棒技术。假激酶是激酶样蛋白,其最近作为信号转导的关键调节剂出现,因此可以代表一类新型的药物靶标。与激酶不同,假激酶的催化效率相当差或不存在,使得难以解析其核苷酸结合位点的功能。已证明基于热变性的方法是确定与纯化的假激酶的配体结合能力的有力方法,并且可以告知核苷酸结合位点的潜在可药物性。该测定利用了当荧光染料(在这种情况下为SYPROOrange)与当蛋白质经历热变性时暴露的疏水性贴片结合时产生的荧光变化。已知配体结合蛋白质增加其热稳定性,其通过在未配对蛋白质和配体蛋白质之间的热变性曲线中观察到的移动来反映。这一广义协议也可以适合于其他蛋白家族。此外,基于热变性的方法可用于鉴定可提高蛋白质稳定性的最佳缓冲液条件

材料和试剂

  1. 纯化的蛋白质(储备溶液优选浓度高于20μM)(Murphy等人,2013)
  2. 1μM二硫苏糖醇(DTT)储液(Astral Scientific,目录号:C-1029)
  3. DMSO(高级)(Sigma-Aldrich,目录号:D-1435)
  4. MilliQ水
  5. 核苷酸溶液(在20mM Tris中制备的10mM储备液)(pH 8)
    1. ATP(Sigma-Aldrich,目录号:A2383)
    2. ADP(Sigma-Aldrich,目录号:A2754)
    3. AMP-PNP(Sigma-Aldrich,目录号:A2647)
    4. GTP(Sigma-Aldrich,目录号:G8877)
  6. 二价阳离子盐溶液(50mM储备液,在MilliQ水中)
    1. MnCl 2(Sigma-Aldrich,目录号:203734)
    2. MgCl 2(Sigma-Aldrich,目录号:M8266)
  7. 激酶抑制剂溶液(100mM DMSO中的2mM储备液)(例如泛激酶抑制剂,Staurosporine,Sigma-Aldrich,目录号:S5921)
  8. 热漂移测定缓冲液(参见配方)
  9. SYPRO Orange(Sigma-Aldrich,目录号:S5692)(参见配方)

设备

  1. RT-PCR管GST-RG01(Gene Targets Solutions)
  2. 1.5ml微量离心管
  3. Qiagen/Corbett Rotor-Gene 3000 RT-PCR机器(QIAGEN)(Murphy等人,2013)

软件

  1. Microsoft Excel或Prism

程序

  1. 热迁移测定(TSA)测试运行以测定蛋白质的最佳量
    1. 准备您的蛋白质样品在缓冲液中的稀释系列从1   至10μM的最终浓度,总体积为25μl。 加入1μl的1x   SYPRO ®橙色到每个管
    2. 使用进行热循环仪运行   参数如步骤3所述。分析熔体曲线(参见步骤   4),并确定给出至少30的蛋白质的最佳量 荧光单元。 如果荧光信号减少蛋白质的量 饱和。
      注意:典型的最终蛋白质浓度为约5 μM。 Rotor-Gene 3000配备  具有增益优化功能 可以调整为最佳信号(参考制造商的 手动)。
  2. 测定
    1. 首先定义您需要测试的条件数, 包括适当的控制,如蛋白质样品在缓冲液中,如果 测试核苷酸结合,或在DMSO的存在下,如果测试化合物。   这将确定蛋白质所需的总混合物的总体积   进行整个实验。 每个反应总共进行 体积为25μl。
    2. 准备在冰上,只是之前使用主混合 含有您的蛋白质在热漂移测定缓冲液中使用 产生峰值荧光的蛋白质浓度(无信号   饱和度)。
    3. 分配23微升蛋白质/缓冲液,加1   μl的配体(10至200μM)或1μl的DMSO /缓冲液作为对照 实验和1μl稀释的SYPRO(R)Orange(1:100)。 准备管 重复。 如果要测试的蛋白质在室内高度不稳定 温度,建议在冰上准备管。 核苷酸 溶液通常以200μM的终浓度使用, 最终为1mM的二价阳离子和最终为40μM的激酶抑制剂。
  3. 热循环程序
    1. 基于荧光的热漂移测定可以使用 结合样品温度控制和染料的仪器 荧光检测。 在这种情况下,我们使用Qiagen/Corbett Rotor-Gene 3000 RT-PCR机。
    2. 从25℃开始,保持在25℃   持续2分钟,以1℃/min的温度升高65个循环(25℃至90℃) °C),每°C读取荧光强度。 返回到25°C。 励磁   是在470nm(绿色通道),发射是在555nm(黄色通道)。
  4. 数据分析
    1. 将数据保存为荧光强度与温度。 导出文件中的 格式适合导入到Microsoft Excel或Prism进行分析
    2. 最高荧光密度用作数据的截止值, 因此在高荧光密度峰后去除所有数据。 适合 荧光强度曲线与使用Prism的Boltzmann S形曲线。
    3. 获得蛋白质的熔解温度(T m) 缓冲液/DMSO,其对应于蛋白质解折叠的中点 曲线。 类似地,当加入配体时获得蛋白质的T m。
    4. 计算ΔTsub = T sub配体-T sub缓冲液/DMSO。 正ΔTm表示 配体稳定蛋白质免于变性,因此结合   蛋白质。 值≥2-3℃是配体结合的指标。
    5. 以下是典型的TSA实验的示例。
      注意: 如果PRISM软件不可用,则非线性回归分析 实验数据可以使用Microsoft Excel电子表格进行 (Brown,2001)。


    图1. TSA实验的实例。(顶部)在存在和不存在核苷酸的情况下获得小鼠MLKL的热迁移测定数据。 在不存在配体的情况下,解折叠转变的中点被观察到为44.8℃,而在ATP存在下T m移动到53.8℃,诱导9℃的位移。 (底部)总结在ATP,GTP和AMPPNP存在下对mMLKL获得的T m和ΔTm值的表。

食谱

  1. 热移动分析缓冲区
    可以根据每种蛋白质来优化用于稀释蛋白质的缓冲液的组成。 以下配方用于研究假性激酶 20mM Tris(pH8)
    150mM NaCl 1 mM DTT
  2. SYPRO ®橙色
    在100%高等级DMSO中1至100稀释

笔记

  1. 虽然单个结构域蛋白的热变性主要导致单个过渡阶段,多结构域蛋白可以展现不同的过渡态。 然而,如果配体紧密结合到一个结构域,其可以导致热变性曲线的变化,提供用于测定配体结合的易处理方法。 还存在配体可以紧密结合但不诱导T m1变化的情况。
  2. 所描述的方案已经适应于Qiagen/Corbett Rotor-Gene 3000 RT-PCR机。每次实验所需的蛋白质量和SYPRO橙色稀释液可能需要根据所使用的RT-PCR机器进行调整。

致谢

我们感谢蒙纳士大学蛋白生产单位访问用于热迁移测定开发的Corbett RT-PCR仪器。这项工作得到国家卫生和医学研究委员会(NHMRC)资助(1016647,461221,1016701,637342,1025594,1046984)和与J.M.H.,N.A.N.的研究金的支持。和J.S。澳大利亚研究委员会与P.E.C.,J.J.B.和J.M.M。以及来自澳大利亚癌症研究基金,维多利亚州政府运营基础设施支持和NHMRC IRIISS拨款(361646)的额外支持。该测定是在完成以下研究的过程中开发的:Murphy等人(2013)和Murphy等人(2014)。

参考文献

  1. Brown,A.M。(2001)。 使用Microsoft Excel电子表格对实验数据进行非线性回归分析的分步指南。 Comput 方法程序Biomed 65(3):191-200。
  2. James,M.M.,Isabelle,S.L.,Joanne,M.H.,Maria,C.T.,Samuel,N.Y.,Pooja,S.,Guillaume,L.,Warren,S.A.,Jeffrey,J.B.and John, 了解人类和小鼠MLKL的晶体结构所显示的假性激酶之间不同核苷酸结合机制的演变。 Biochem J ,457(3):369-377。
  3. 本发明的另一个方面涉及一种用于治疗和/或预防糖尿病的方法,所述方法包括以下步骤Varghese,LN,Tannahill,GM,Hatchell,EC,Majewski,IJ,Okamoto,T.,Dobson,RC,Hilton,DJ,Babon,JJ,Nicola,NA,Strasser,A.,Silke, (2013年)。 假性激酶MLKL介导坏死病通过分子开关机制。 免疫 39(3):443-453。
  4. Murphy,JM,Zhang,Q.,Young,SN,Reese,ML,Bailey,FP,Eyers,PA,Ungureanu,D.,Hammaren,H.,Silvennoinen,O.,Varghese,LN,Chen,K.,Tripaydonis ,A.,Jura,N.,Fukuda,K.,Qin,J.,Nimchuk,Z.,Mudgett,MB,Elowe,S.,Gee,CL,Liu,L.,Daly,RJ,Manning, ,Babon,JJ和Lucet,IS(2014)。 一种基于其核苷酸结合性质将假性激酶分类的可靠方法。 Biochem J 457(2):323-334。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lucet, I. S., Hildebrand, J. M., Czabotar, P. E., Zhang, J., Nicola, N. A., Silke, J., Babon, J. J. and Murphy, J. M. (2014). Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay. Bio-protocol 4(11): e1135. DOI: 10.21769/BioProtoc.1135.
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