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Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST)
通过微量热泳法(MST)测定FNR-NrdI的相互作用   

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Abstract

This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus (Lofstad et al., 2016). NrdI is essential in the activation of the manganese-bound form of the class Ib ribonucleotide reductase (RNR) system. RNRs, in turn, are the only source of the de novo synthesis of deoxyribonucleotides required for DNA replication and repair in all living organisms.

Keywords: MST(MST), Microscale thermophoresis(微量热泳法), Protein-protein interaction(蛋白-蛋白相互作用), KD(KD), Binding constant(结合常数)

Background

Protein-protein interactions are often characterised in terms of the associated dissociation constant, KD. The binding constant can be established using a variety of techniques, such as isothermal calorimetry (ITC), NMR spectroscopy, and surface plasmon resonance (SPR). An alternative method is based on thermophoresis, a phenomenon where distinct molecules (such as a protein-protein complex versus individual proteins) respond differently to a temperature gradient (Duhr and Braun, 2006; Seidel et al., 2013). This method is rapid, no sample immobilisation is needed and the sample requirement is low. Briefly, one of the proteins is labelled with a fluorescent dye and kept at a constant, low concentration. A dilution series is set up, where the other protein is diluted up to 16 times, creating a vast concentration range. The two proteins are subsequently mixed and loaded into capillaries, which are scanned in the MonolithTM NT.115 instrument, developed and sold exclusively by NanoTemper. The samples are subjected to a temperature gradient, and the movement of the fluorescently labelled molecule is tracked. The difference in the fluorescence of the molecule at the initial temperature and at the new temperature is used to generate a binding curve as a function of the concentration of the unlabelled protein.

Materials and Reagents

  1. Tubes
    15 (VWR, catalog number: 525-0400 )
    50 ml (VWR, catalog number: 525-0402 )
  2. Pipette tips  
    0.1-10 μl (VWR, catalog number: 613-0735 )
    1-200 μl (VWR, catalog number: 613-0740 )
    100-1,250 μl (VWR, catalog number: 613-0739 )
  3. 0.2 μm filter (SARSTEDT, catalog number: 83.1826.001 )
  4. Syringe, 50 ml (BD, catalog number: 300865 )
  5. Protein 1 (here, NrdI, locus-tag: bc1353) and protein 2 (here, FNR1 bc0385, FNR2 bc4926, FNR3 bc1495)
  6. MO-L001 MonolithTM Protein Labeling Kit RED-NHS (Amine Reactive) (NanoTemper) (contains NT-647-NHS dye [store at -20 °C]; spin column for buffer exchange [store at 4 °C]; gravity flow column for purification [store at 4 °C]; labeling buffer [store at 4 °C])
  7. MO-K002 MonolithTM NT.115 Standard Treated Capillaries (NanoTemper) (contains MonolithTM NT.115 Standard Treated Capillaries [store at RT]; 10% Tween 20 [store at 4 °C]; Albumin fraction A [store at 4 °C]; MST buffer [store at -20 °C]; 200 μl vials)
  8. DMSO (Sigma-Aldrich, catalog number: D4540 )
  9. Liquid N2
  10. HEPES (AppliChem, catalog number: A3724 )
  11. Potassium chloride (KCl) (EMD Millipore, catalog number: 104936 )
  12. Tween-20
  13. Buffer A (see Recipes)

Equipment

  1. Pipettes (various sizes)
  2. Benchtop centrifuge
  3. Vortex
  4. MonolithTM NT.115 with Blue/Red filter (NanoTemper Technologies, model: MO-G008 )

Procedure

  1. Labelling the protein with fluorescent dye
    1. The instructions for this step are based on using a dye and consumables supplied from NanoTemper. The NanoTemper dyes (namely RED, BLUE, and GREEN) are available with either an NHS-ester group that reacts with primary amines on for example lysines, or a maleimide group that reacts with sulfhydryl groups on e.g., cysteines. Other dyes may also be used, or alternatively proteins that have a fluorescent tag, in which case no further labelling should be required. Note that your dye (or tag) of choice must be compatible with the filters that are installed in the instrument and that the dye, once bound to the protein, should not block any potential binding sites.
      Note: When labelling one of your proteins with a fluorescent dye, you first need to determine which of the two proteins you want to label. It may be useful to consider that you will only use a very small amount of the labelled protein and that, depending on the KD, you might have to use much more of the unlabelled protein (at least 20x the KD). Our measured binding affinities were in the 20-50 μm range and in order to have a satisfying binding curve the protein concentrations in the capillaries were approximately 20 nM of labelled NrdI, and 1 mM FNR in capillary 1 to 15 nM in capillary 16 with a 1:1 dilution series.
    2. We chose to label NrdI since we were testing its binding affinity to three other proteins (FNRs). NrdI was labelled with the RED-NHS dye, as it contains eight lysines and no cysteines. Based on the NrdI crystal structure (Røhr et al., 2010) we do not expect any of the lysines to be in the proximity of the binding site.
      Note: We have used buffer A (see Recipes) as our labelling buffer. When using the NHS-based dyes, the protein should not be in a buffer that contains primary amines, as these might compete with the protein in the labelling reaction. Similarly, when using the maleimide-based dyes, the protein cannot be in a buffer that contains sulfhydryl groups. Also, the reducing agents DTT and β-mercaptoethanol should not be used with either dye (TCEP may be used for both dyes). If the protein is dissolved in a buffer that contains any of the above-mentioned reagents, a buffer exchange step is required prior to labelling. NanoTemper provides a labelling buffer in the labelling kit that may be used, as well as a spin column for buffer exchange.
      1. Dilute the protein stock to a concentration that is lower than your expected KD using labelling buffer. NrdI was diluted to 25 μM and a final volume of 188 μl.
        Note: NanoTemper recommends using a protein concentration between 2 and 20 μM and a volume of 100 μl.
      2. Dissolve the dye in 100% DMSO (30 μl). Vortex well and ensure that all the dye is dissolved.
      3. Dilute the dye with labelling buffer to a concentration that is 2-3x the concentration of your protein. Here, the NT-647 dye was diluted to 75 μM and a final volume of 188 μl.
      4. Mix the protein and the dye solution in a 1:1 volume ratio and incubate at room temperature in the dark for 30 min.
        Note: This is the suggested reaction condition as described in the NanoTemper labeling kit manual, but varying the dye:protein ratio or the incubation time might increase the labeling yield.
      5. In the meantime, wash the gel filtration column that is supplied in the kit. Pour off the storage solution and equilibrate the column with 3 x 3 ml buffer A using gravity flow.
      6. Add the protein/dye mix to the centre of the resin in the column. When the sample has completely entered the column bed, add buffer to adjust the total volume of applied sample to 500 μl (We added 500 - (2 x 188) = 124 μl buffer). Do not collect the flow through.
      7. Elute the labelled protein with 600 μl buffer A and collect the flow through in a clean tube.
        Note: It is also possible to collect smaller fractions (e.g., 4 x 150 μl) and check the fluorescence intensity of each fraction in order to find the fraction that contains the labelled protein. Note that free dye might elute at the end.
      8. Perform a capillary scan (cap scan) and check the fluorescence intensity of the labelled protein.
        Note: The degree of labelling should also be determined (see Notes).
      9. Aliquot the protein in 20 μl fractions, flash freeze in liquid N2 and store at -80 °C. The fluorescently labelled NrdI protein was used within 4 months.

  2. KD-measurement
    1. Spin down protein stocks at 15,000 x g for 5 min.
    2. Dilute the fluorescently labelled protein with buffer A to a concentration that gives a fluorescence count between 200 and 1,500 (the detection limits of the instrument). Remember that the protein will be diluted 1:1 in the actual experiment and that you might have to increase the concentration of the labelled protein to remain within the detection limit. The fluorescently labelled NrdI protein was diluted 1:25.
      Note: You can adjust the counts by either changing the LED-power or the concentration of your fluorescently labelled molecule.
    3. Perform a cap scan to ensure that the concentration is within the detection limits and that the protein is not sticking to the capillary walls (sticking is evidenced by shoulders or double peaks in the cap scan traces instead of a smooth, symmetrical curve) (Figure 1).
      Note: If the protein is sticking to the capillaries, you may want to try adding detergents or additives such as Tween-20, BSA, or Pluronic F-127, changing the pH or ionic strength of your buffer, changing to a different buffer, or using the premium coated capillaries.


      Figure 1. Cap scan of 1:25 dilution NrdI in standard capillaries at 20% LED-power

    4. Number 16 microtubes 1-16.
      Note: The tubes are provided with the capillaries.
    5. Pipet 10 μl buffer A in tubes 2-16.
      Note: Use a 10 μl pipette in steps B5, B6, and B7 to improve accuracy.
    6. Pipet 20 μl of the unlabelled protein into tube 1. Remove 10 μl, and add these to tube 2. Mix by pipetting up and down several times. Remove 10 μl and add these to tube 3. Mix well. Continue like this for all the tubes. In tube 16, remove 10 μl and discard the liquid. You should now have 10 μl in all the tubes.
      Note: The unlabelled protein must also be in the assay buffer to avoid any buffer dilution effects. You may want to use a new pipet tip for each transfer to ensure consistent volumes in the serial dilution.
    7. Add 10 μl of the fluorescently labelled protein to all the tubes and mix well. Use a new pipet tip each time.
      Note: Do not use less than 20 μl total volume of reaction mixture so that sticking and evaporation issues are minimised and pipetting errors are reduced.
    8. Incubate the reaction mixtures for 5 min at room temperature.
    9. Load the samples into the capillaries and place the capillaries in the sample holder.
      Note: Be careful not to touch the capillaries in the middle and avoid any bubbles in the middle of the capillary.
    10. Put the sample holder in the MST-instrument. Perform a cap scan of all the capillaries and run the MST-experiment. The NrdI-FNR interaction was measured using 20% LED-power and 40% MST-power.
      Note: The LED-power can be adjusted up to 95%, whereas the MST-power should ideally not be increased to over 40%.

Data analysis

  1. Inspecting the raw data
    1. Evaluate the cap scans. They should be symmetrical and should not exhibit any bumps in the traces. The maximum fluorescence intensity of each peak should not differ by more than ± 10% (Figure 2).
      Note: If the fluorescence differs by more than 10% in a concentration-dependent manner, it either indicates that the fluorescence is changing upon binding, or that the labelled protein is lost due to precipitation or unspecific adsorption during the sample preparation, which is carried through in the serial dilution. If the fluorescence differs by more than 10% in a non-concentration dependent manner, the assay conditions must be optimised, as described in step B3.


      Figure 2. Cap scan inspection. A. Cap scan of a representative experiment; B. Variation in fluorescence intensity of the cap scan in A.

    2. Inspect the MST-traces (Figure 3). Any bumps indicate that the samples are not homogenous. The traces should also not cross each other.


      Figure 3. MST traces of a representative experiment

      Note: Thermophoresis can be both positive and negative. We have observed positive thermophoresis (see Figure 3), i.e., a net decrease in the observed fluorescence because the fluorescently labelled molecules move from the hotter region in the capillaries to the cooler region. We attribute the increase in the MST-traces at the end of each run to convection (Figure 3).

  2. Data processing and analysis
    Data processing and analysis were carried out using the MO.Affinity Analysis program (NanoTemper). Three or four replicates were merged to form one dataset.
    1. Select the appropriate binding model. We selected KD.
    2. Enter the concentration of your fluorescently labelled molecule (TargetConc) and fix this by ticking the box.
    3. The program will compute the KD-value and the associated uncertainty. The NrdI-FNR interaction was analysed using the ‘Thermophoresis with T jump’ evaluation strategy.
      Note: The program automatically computes the binding constant taking into account both temperature jump (immediate change in fluorescence upon the temperature change) and thermophoresis effects (the movement of the molecules in the temperature gradient). This mode is called Thermophoresis with T jump. It is possible to change the evaluation strategy by selecting the ‘Expert mode’. In this mode four options are available: Thermophoresis with T jump; Thermophoresis; T jump; and Manual. In the manual mode you can manually define the before/after regions that are to be used in the analysis. Further explanation and discussion of the different modes can be found in Scheuermann et al. (2016).
    4. Inspect the binding curve. The amplitude of the curve should be at least 5 response units and the noise in the baseline should be at least 3 times less than the amplitude.
    5. To compare several datasets, the fluorescence of each dataset may be normalised, △Fnorm, by selecting this option in the MO.Affinity Analysis program. The normalised fluorescence of each dataset can, in turn, be divided by the amplitude of the binding curve, as seen in Figure 4. We transferred the data points and the associated uncertainties as well as the fitted traces from the MO.Affinity Analysis program to Origin, where the △Fnorm-values were divided by the amplitude of the binding curve and the curves were re-plotted (Figure 4). 


      Figure 4. Normalised fluorescence traces (adapted from Lofstad et al., 2016)

Notes

  1. The binding constants may vary from one protein preparation to another.
  2. Valuable control experiments that should be carried out include testing the binding affinities at different pH-values and at different ionic strengths, and also testing the fluorescently labelled protein against either a known non-binding protein or against a protein that is known to bind strongly to the labelled protein (or both).
  3. Determining the degree of labelling (as described in the FAQ: How do I determine the protein concentration after labeling and the degree of labeling (DOL)? [NanoTemper]):
    1. Collect an absorbance spectrum of the labelled protein using a UV-vis spectrophotometer.
    2. Read the absorbance at 280 nm and the absorbance at λmax of the dye (varies depending on the dye used).
    3. The concentration of the labelled protein can be found using the following formula:



      where,
      d is the path length of the spectrophotometer,
      εprot is the extinction coefficient of the protein,
      Amax is the absorbance at λmax,
      CF is the correction factor of the dye, taking into account the absorption of the dye at 280 nm.
      The degree of labelling can be found using the equation below:



      where,
      εmax is the extinction coefficient of the dye.

Recipes

  1. Buffer A (50 mM HEPES, 100 mM KCl, 0.05% Tween-20)
    1.192 g HEPES
    0.745 g KCl
    Make up to 90 ml with Milli-Q distilled H2O, adjust the pH to 7.5 using KOH, and make up to final volume 100 ml with Milli-Q distilled H2O. If necessary, adjust the pH again
    Sterile filter the solution using a syringe and 0.22 μm filter and store at 4 °C until use (the buffer has been used within 5 days)
    Add 500 μl 10% Tween-20 to a final concentration of 0.05% prior to the experiments

Acknowledgments

This protocol is based on the user manual supplied with the MonolithTM NT.115 instrument (NanoTemper) and the FAQ: How do I determine the protein concentration after labeling and the degree of labeling (DOL)? (NanoTemper). This work is funded by the Norwegian Research Council (Projects 231669 and 214239). The MST instrument is operated with the financial support of the South-Eastern Norway Regional Health Authority (Grant 2015095; Regional Core Facility for Structural Biology). A brief description of this protocol has previously been published in Lofstad et al. (2016).

References

  1. Duhr, S. and Braun, D. (2006). Why molecules move along a temperature gradient. Proc Natl Acad Sci U S A 103(52): 19678-19682.
  2. Lofstad, M., Gudim, I., Hammerstad, M., Rohr, A. K. and Hersleth, H.-P. (2016). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus. Biochemistry 55(36): 4998-5001.
  3. Røhr, Å. K., Hersleth, H.-P. and Andersson, K. K. (2010). Tracking flavin conformations in protein crystal structures with Raman spectroscopy and QM/MM calculations. Angew Chem Int Ed Engl 49(13): 2324-2327.
  4. Scheuermann, T. H., Padrick, S. B., Gardner, K. H. and Brautigam, C. A. (2016). On the acquisition and analysis of microscale thermophoresis data. Anal Biochem 496: 79-93.
  5. Seidel, S. A., Dijkman, P. M., Lea, W. A., van den Bogaart, G., Jerabek-Willemsen, M., Lazic, A., Joseph, J. S., Srinivasan, P., Baaske, P., Simeonov, A., Katritch, I., Melo, F. A., Ladbury, J. E., Schreiber, G., Watts, A., Braun, D. and Duhr, S. (2013). Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59(3): 301-315.

简介

该协议描述了如何使用Monolith TM NT.115仪器(NanoTemper)通过微量热泳法(MST)测量蛋白质 - 蛋白质相互作用。我们已经使用方案来确定三种不同的黄素氧还蛋白还原酶(FNR)和来自蜡样芽孢杆菌(Lofstad等)的黄素氧还蛋白样蛋白NrdI之间的结合亲和力, 2016)。 NrdI对于Ib类核糖核苷酸还原酶(RNR)系统的锰结合形式的活化至关重要。反过来,RNRs是所有生物体中DNA复制和修复所需的脱氧核糖核苷酸合成的唯一来源。

蛋白质 - 蛋白质的相互作用通常以相关的解离常数(K D )为特征。可以使用诸如等温量热法(ITC),NMR光谱和表面等离子体共振(SPR)的各种技术来建立结合常数。另一种方法是基于热泳法,其中不同分子(例如蛋白质 - 蛋白质复合物与单个蛋白质)对温度梯度的反应不同(Duhr和Braun,2006; Seidel等人, 2013)。该方法快速,不需要样品固定,样品要求较低。简言之,其中一种蛋白质用荧光染料标记并保持在恒定的低浓度。建立稀释系列,其他蛋白质稀释至16倍,产生广泛的浓度范围。随后将两种蛋白质混合并加载到毛细管中,毛细管在Monolith TM NT.115仪器中扫描,由NanoTemper专门开发和销售。对样品进行温度梯度,并跟踪荧光标记的分子的移动。在初始温度和新温度下分子荧光的差异用于产生作为未标记蛋白质浓度的函数的结合曲线。

关键字:MST, 微量热泳法, 蛋白-蛋白相互作用, KD, 结合常数

材料和试剂

  1. 管子
    15(VWR,目录号:525-0400)
    50毫升(VWR,目录号:525-0402)
  2. 移液器提示
    0.1-10μl(VWR,目录号:613-0735)
    1-200μl(VWR,目录号:613-0740)
    100-1,250μl(VWR,目录号:613-0739)
  3. 0.2μm过滤器(SARSTEDT,目录号:83.1826.001)
  4. 注射器,50ml(BD,目录号:300865)
  5. 蛋白质1(这里,NrdI,基因座标签:bc1353 )和蛋白质2(这里,FNR1 > bc0385 ,FNR2bc4926 ,FNR3 < bc1495 )
  6. 蛋白标记试剂盒RED-NHS(Amine Reactive)(NanoTemper)(含有NT-647-NHS染料[-20℃储存];用于缓冲液交换的离心柱[存储在4℃];用于纯化的重力流柱[在4℃下储存];标记缓冲液[在4℃下储存])
  7. MO-K002 Monolith TM NT.115标准处理毛细管(NanoTemper)(包含Monolith TM NT.115标准处理毛细管[RT]; 10%Tween 20 [在4℃];白蛋白部分A [在4℃下储存]; MST缓冲液(-20℃储存);200μl小瓶)
  8. DMSO(Sigma-Aldrich,目录号:D4540)
  9. 液体N 2
  10. HEPES(AppliChem,目录号:A3724)
  11. 氯化钾(KCl)(EMD Millipore,目录号:104936)
  12. Tween-20
  13. 缓冲液A(参见食谱)

设备

  1. 移液器(各种尺寸)
  2. 台式离心机
  3. 涡流
  4. 具有蓝/红滤光片的MonolithTM NT.115(NanoTemper Technologies,型号:MO-G008)

程序

  1. 用荧光染料标记蛋白质
    1. 该步骤的说明基于使用由NanoTemper提供的染料和消耗品。 NanoTemper染料(即,RED,BLUE和GREEN)可用与例如赖氨酸的伯胺反应的NHS-酯基团或与例如上的巯基反应的马来酰亚胺基团。 ,半胱氨酸。也可以使用其它染料,或者使用具有荧光标签的蛋白质,在这种情况下不需要进一步标记。请注意,您选择的染料(或标签)必须与仪器中安装的过滤器兼容,并且一旦与蛋白质结合的染料不应阻挡任何潜在的结合位点。
      注意:当用荧光染料标记您的蛋白质之一时,您首先需要确定要标记的两种蛋白质中的哪一种。考虑到您只能使用非常少量的标记蛋白,这可能是有用的,根据K ,您可能需要使用更多的未标记蛋白质(至少20x K D )。我们测量的结合亲和力在20-50μM范围内,为了具有令人满意的结合曲线,毛细管中的蛋白质浓度为约20nM的标记NrdI,毛细管1中的1mM FNR在毛细管16中具有1:1稀释系列。
    2. 我们选择标记NrdI,因为我们正在测试其与三种其他蛋白质(FNR)的结合亲和力。 NrdI标有RED-NHS染料,因为它含有8个赖氨酸,没有半胱氨酸。基于NrdI晶体结构(Røhr等人,2010),我们不期望任何赖氨酸位于结合位点附近。
      注意:我们已经使用了缓冲区A(见配方)作为标签缓冲区。当使用基于NHS的染料时,蛋白质不应该在含有伯胺的缓冲液中,因为它们可能与标记反应中的蛋白质竞争。类似地,当使用马来酰亚胺类染料时,蛋白质不能在含巯基的缓冲液中。此外,还原剂DTT和β-巯基乙醇不应与任一染料一起使用(TCEP可用于两种染料)。如果蛋白质溶解在含有任何上述试剂的缓冲液中,则在标记之前需要缓冲液交换步骤。 NanoTemper在可能使用的标签套件中提供标签缓冲液,以及用于缓冲液交换的自旋柱。
      1. 使用标记缓冲液将蛋白质原料稀释至低于您预期的> D 的浓度。 NrdI稀释至25μM,最终体积为188μl 注意:NanoTemper建议使用2至20μM的蛋白质浓度和100μl的体积。
      2. 将染料溶解于100%DMSO(30μl)中。旋转良好,确保所有染料溶解
      3. 将染料溶解于100%DMSO(30μl)中。旋转良好,确保所有染料溶解
      4. 用标记缓冲液稀释染料,浓度为蛋白质浓度的2-3倍。在这里,将NT-647染料稀释至75μM,最终体积为188μl
      5. 以1:1体积比混合蛋白质和染料溶液,并在室温下在黑暗中孵育30分钟。
        注意:这是NanoTemper标签套件手册中描述的建议的反应条件,但改变染料:蛋白质比例或孵育时间可能会增加标签产量。
      6. 同时,洗涤试剂盒中提供的凝胶过滤柱。倒出储存溶液,并用3×3ml缓冲液A使用重力流平衡柱。
      7. 将蛋白质/染料混合物加入柱中树脂的中心。当样品完全进入柱床时,加入缓冲液以将应用样品的总体积调节至500μl(我们添加500-(2×188)=124μl缓冲液)。不要收集流经。
      8. 用600μl缓冲液A洗脱标记的蛋白质,并在干净的管中收集流动 注意:也可以收集较小的级分(例如4×150μl)并检查每个级分的荧光强度,以便找到含有标记蛋白质的级分。请注意,免费染料可能会在最后洗脱。
      9. 进行毛细血管扫描(帽扫描)并检查标记蛋白的荧光强度。
        注意:还应确定标签的程度(见注释)。
      10. 将蛋白质等分成20μl级分,在液体N 2中快速冷冻并储存在-80℃。 4个月内使用荧光标记的NrdI蛋白
  2. D - 测量
    1. 将蛋白质原料转入15,000 x g,持续5分钟
    2. 用缓冲液A稀释荧光标记的蛋白质,使荧光计数在200和1,500之间(仪器的检测限)。请记住,实际实验中蛋白质将以1:1稀释,并且您可能必须增加标记蛋白质的浓度以保持在检测限。荧光标记的NrdI蛋白以1:25稀释 注意:您可以通过更改LED功率或荧光标记分子的浓度来调整计数。
    3. 进行帽扫描以确保浓度在检测范围内,并且蛋白质不粘附到毛细管壁(粘贴由帽扫描轨迹中的肩峰或双峰证实,而不是平滑对称的曲线)(图1 )。
      注意:如果蛋白质粘附到毛细血管上,您可能需要尝试添加去垢剂或添加剂,如Tween-20,BSA或Pluronic F-127,改变缓冲液的pH或离子强度,改为不同的缓冲液,或使用优质涂层毛细管。


      图1. 20%LED电源标准毛细管中1:25稀释度NrdI的Cap扫描

    4. 16号微管1-16。
      注意:管上装有毛细管。
    5. 吸管10-16缓冲液A在管2-16。
      注意:在步骤B5,B6和B7中使用10μl移液器可提高准确度。
    6. 将20μl未标记的蛋白质吸管至管1中。取出10μl,并将其加入到管2中。通过上下移动多次混合。取出10μl,加入管3中。混匀。继续这样的所有管。在管16中,取出10μl,弃去液体。你现在应该在所有的管子里有10μl。
      注意:未标记的蛋白质也必须在测定缓冲液中以避免任何缓冲液稀释效应。您可能需要为每次传输使用新的移液器提示,以确保连续稀释时的体积一致。
    7. 将10μl荧光标记的蛋白质加入所有管中,并充分混匀。每次使用新的吸头。
      注意:不要使用少于20μl的总体积的反应混合物,以便使粘附和蒸发问题最小化,并且减少移液误差。
    8. 将反应混合物在室温下孵育5分钟
    9. 将样品装入毛细管并将毛细管置于样品架中。
      注意:注意不要碰触中间的毛细血管,并避免毛细血管中间有任何气泡。
    10. 将样品架置于MST仪器中。对所有毛细管进行帽扫描,并运行MST实验。使用20%LED功率和40%MST功率测量NrdI-FNR相互作用。
      注意:LED功率可以调整高达95%,而MST功率理想地不会增加到40%以上。

数据分析

  1. 检查原始数据
    1. 评估上限扫描。它们应该是对称的,并且不应该在痕迹中表现出任何颠簸。每个峰的最大荧光强度不应大于±10%(图2) 注意:如果荧光以浓度依赖的方式差异超过10%,则表明荧光在结合时发生变化,或标记的蛋白质在样品制备过程中由于沉淀或非特异性吸附而损失,其通过连续稀释进行。如果荧光以非浓度依赖的方式差异超过10%,则必须优化测定条件,如步骤B3所述。


      图2. Cap扫描检查 A.代表性实验的Cap扫描; B.在A.扫描中的荧光强度的变化
    2. 检查MST-trace(图3)。任何颠簸表示样品不均匀。痕迹也不能互相交叉。


      图3.代表性实验的MST跟踪

      注意:热泳可以是阳性和阴性。我们已经观察到阳性热泳(见图3),即观察到的荧光的净减少,因为荧光标记的分子从毛细血管中较热的区域移动到较冷的区域。我们将每次运行结束时的MST跟踪的增加归因于对流(图3)。

  2. 数据处理与分析
    使用MO.Affinity Analysis程序(NanoTemper)进行数据处理和分析。三或四个重复合并形成一个数据集。
    1. 选择适当的绑定模型。我们选择了 K D
    2. 输入荧光标记分子(TargetConc)的浓度,并通过勾选方框来解决这个问题。
    3. 该程序将计算 K D 值和相关的不确定性。 NrdI-FNR的相互作用使用"热泳与T跳"评估策略进行分析 注意:程序自动计算结合常数,同时考虑温度跳变(温度变化时荧光的立即变化)和热泳效应(分子在温度梯度中的移动)。这种模式称为带T跳的热泳。通过选择"专家模式"可以改变评估策略。在这种模式下,有四个选项可供选择:带T跳的热泳热泳T跳;和手动。在手动模式下,您可以手动定义要在分析中使用的前/后区域。不同模式的进一步解释和讨论可以在Scheuermann等人(2016)。
    4. 检查绑定曲线。曲线的幅度应至少为5个响应单位,基线中的噪声应至少比振幅小3倍。
    5. 为了比较几个数据集,可以通过在MO.Affinity Analysis程序中选择此选项来将每个数据集的荧光归一化为△ norm 。如图4所示,每个数据集的归一化荧光又可以除以结合曲线的幅度。我们将数据点和相关不确定度以及MO.Affinity分析程序中的拟合轨迹转移到原产地,其中△ norm - 值除以结合曲线的幅度,并重新绘制曲线(图4)。

      图4.归一化荧光迹线(改编自Lofstad等人,2016)

笔记

  1. 结合常数可以从一种蛋白质制剂到另一种不同。
  2. 应进行的有价值的对照实验包括测试不同pH值和不同离子强度下的结合亲和力,并测试荧光标记的蛋白质与已知的非结合蛋白质或已知与蛋白质强烈结合的蛋白质标记的蛋白质(或两者)。
  3. 确定标签的程度(如FAQ中所述:如何确定标记后的蛋白质浓度和标记度(DOL)?[NanoTemper]):
    1. 使用UV-vis分光光度计收集标记蛋白质的吸收光谱
    2. 读取280nm处的吸光度和染料的λmax 处的吸光度(根据所用的染料而变化)。
    3. 标记蛋白质的浓度可以使用以下公式找到:



      其中,
      d是分光光度计的路径长度,
      ε是蛋白质的消光系数,
      最大值是λmax最大值的吸光度,
      考虑到染料在280nm处的吸收,CF是染料的校正因子 标签的程度可以通过以下公式得到:



      其中,
      ε 是染料的消光系数

食谱

  1. 缓冲液A(50mM HEPES,100mM KCl,0.05%Tween-20) 1.192克HEPES
    0.745克KCl
    用Milli-Q蒸馏的H 2 O 2将其调至90ml,用KOH将pH调节至7.5,用Milli-Q蒸馏的H 2/> O。如有必要,再次调整pH值
    使用注射器和0.22μm过滤器对溶液进行无菌过滤,并在4℃下储存直至使用(缓冲液已在5天内使用)
    在实验前添加500μl10%吐温-20至终浓度为0.05%

致谢

该协议基于Monolith TM NT.115仪器(NanoTemper)和常见问题解答提供的用户手册:如何确定标记后的蛋白质浓度和标记度(DOL)? (NanoTemper)。这项工作由挪威研究理事会资助(项目231669和214239)。 MST仪器在挪威东南部地区卫生局(拨款2015095;结构生物学区域核心设施)的财政支持下运作。此协议的简要描述以前已在Lofstad等人公开。 (2016)。

参考文献

  1. Duhr S.和Braun D.(2006)。为什么分子沿着温度梯度移动。Proc Natl Acad Sci USA 103(52):19678-19682。
  2. Lofstad,M.,Gudim,I.,Hammerstad,M.,Rohr,A.K。和Hersleth,H.-P. (2016)。将Ib类核糖核苷酸还原酶活化为在蜡状芽孢杆菌中的黄素氧还蛋白还原酶。生物化学 55(36):4998-5001。
  3. Røhr,Å。 K.,Hersleth,H.-P.和Andersson,KK(2010)。  跟踪黄素构象蛋白质晶体结构,具有拉曼光谱和QM/MM计算。 Angew Chem Int Ed Engl 49(13):2324-2327。
  4. Scheuermann,TH,Padrick,SB,Gardner,KH和Brautigam,CA(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26739938 "target ="_ blank">关于微量热泳法数据的获取和分析。 Anal Biochem 496:79-93。
  5. Seidel,SA,Dijkman,PM,Lea,WA,van den Bogaart,G.,Jerabek-Willemsen,M.,Lazic,A.,Joseph,JS,Srinivasan,P.,Baaske,P.,Simeonov, Katritch,I.,Melo,FA,Ladbury,JE,Schreiber,G.,Watts,A.,Braun,D。和Duhr,S.(2013)。  Microscale thermophoresis量化在以前具有挑战性条件下的生物分子相互作用。 方法59(3) :301-315。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gudim, I., Lofstad, M., Hammerstad, M. and Hersleth, H. (2017). Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST). Bio-protocol 7(8): e2223. DOI: 10.21769/BioProtoc.2223.
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