参见作者原研究论文

本实验方案简略版
Oct 2017
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Isothermal Titration Calorimetry: A Biophysical Method to Characterize the Interaction between Label-free Biomolecules in Solution
等温滴定量热法:一种检测溶液中非标记生物分子相互作用的生物物理方法   

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Abstract

This protocol can be applied to analyze the direct interaction between a soluble protein and a target ligand molecule using Isothermal Titration Calorimetry (ITC, Malvern). ITC allows the biophysical characterization of binding between label-free, non-immobilized and in-solution biomolecules by providing the stoichiometry of the interaction, the equilibrium binding constants and the thermodynamic parameters. ITC monitors heat changes (released and/or absorbed) caused by macromolecular interactions with no restrictions of buffer and molecular weight of the macromolecules.

Keywords: ITC (ITC), Calorimetr (量热法), Macromolecular interaction (大分子互作), Binding affinity (结合亲和力), KAT1 channels (KAT1通道), 14-3-3 proteins (14-3-3蛋白质)

Background

Macromolecular interactions are critical cellular events as they form the basis for signal transduction pathways. Thus, macromolecular interactions are an essential field of research, as they allow a deeper understanding of the molecular mechanisms which underlie both physiological and pathophysiological processes, and the rational design of drugs able to modulate disease-causing macromolecular binding events.

In this context, Isothermal Titration Calorimetry (ITC) is a powerful technique for the characterization of macromolecular interactions. ITC determines the heat change that occurs upon the binding of two molecules. Heat can be absorbed (endothermic reaction) or released (exothermic reaction). ITC monitors such heat changes by determining the differential power, provided by heaters of the instrument to both the reference and the sample cells, needed for counteracting any temperature difference between the two cells during the binding reaction such that no difference in temperature arises between the reference and sample cells (Figure 1).


Figure 1. Principle of Isothermal Titration Calorimetry (ITC). A. Cartoon representation of an isothermal titration calorimetry instrument composed of: a reference cell filled with MilliQ water; a sample cell containing a biomolecule; and an automated injection syringe containing the other binding molecule (ligand) used to titrate the ligand into the sample cell. The sample and reference cells are surrounded by an adiabatic jacket. The system is able to detect temperature differences between the reference and sample cells and to maintain an absence of temperature difference between them (ΔT = 0) by supplying power to both the reference and the sample cell via two heaters. The output of the instrument is the power (μcal/sec) required to maintain ΔT = 0 between the reference and sample cells. B-D. The temperature difference between the reference and the sample cell, induced by the ligand–biomolecule binding, is converted into the power needed to bring the two cells back to the same temperature during the binding reaction. As the titration proceeds, the biomolecule in the sample cell becomes saturated with the ligand, so that less interactions occur and consequently the heat change decreases (C) until the biomolecule is fully saturated and the instrument detects only heat change due to the dilution of the ligand (D).

ITC provides important information about the nature of the macromolecular interaction: the binding stoichiometry (N); the thermodynamic parameters of the binding reaction (enthalpy, ∆H, entropy, ∆S, and Gibbs free energy, ∆G); the strength of the interaction (the equilibrium association constant KA, from which the more commonly used equilibrium dissociation constant KD can be derived).

Among the methods used to characterize macromolecular interactions, ITC has two major advantages: i) the biomolecules are free to move in solution and are not labelled, which insures a direct characterization of the binding event, unbiased by labelling and/or by limitation on molecule motions due to their immobilization on a surface; ii) ITC is the only method that allows a detailed characterization of the binding event by providing not only the binding affinity, but also other critical information, i.e., the binding stoichiometry and the thermodynamic parameters. This information can help significantly in the understanding of the molecular mechanism of the binding reaction, even when no structural data are yet available. Furthermore, they can be used as complementary data to validate structural results.

Recently, I presented crystallographic and functional data showing that the K+ inward rectifier KAT1 (K+ Arabidopsis thaliana 1) channel is regulated by the direct binding of 14-3-3 proteins (Saponaro et al., 2017). In particular, I identified a 14-3-3 mode III binding site at the very C-terminus of KAT1 and co-crystallized it with tobacco 14-3-3 proteins (14-3-3c) to describe the protein complex in atomic detail. The structural results were complemented/supported by measuring, through ITC, the interaction between a synthetic KAT1 C-terminal phosphopeptide (CPP) and 14-3-3c. ITC was employed to quantify the stoichiometry, the equilibrium binding affinity and the thermodynamic parameters of the 14-3-3c-CPP binding reaction.

The aim of this protocol is to provide a detailed description of the setting procedure of an ITC experiment, highlighting the crucial steps and related concerns, and providing, at the same time, a well-established strategy to overcome such problems. Moreover, the present protocol describes the analysis of an ITC measurement of the single binding event in a 14-3-3c/CPP interaction.


Materials and Reagents

  1. Pipette tips (20 μl, 200 μl and 1,000 μl)*
  2. Glass syringe 2.5 ml, 18GA, 8.5IN, PT3 (Malvern Panalytical, catalog number: SYN161714 )
  3. Disposable borosilicate glass tubes, 0.7 ml, 6 x 50 mm (O.D. x Height) (DWK Life Sciences, Kimble Chase, catalog number: 73500-650 )
  4. 0.22 μm vacuum filter*
  5. Purified Nicotiana tabacum 14-3-3c protein recombinantly expressed in E. coli. Protein stock concentration is 100 μM
    Note: The protein was expressed and purified as described in Saponaro et al. (2017).
  6. Synthetic phosphopeptide* (corresponding to the last 5 residues of Arabidopsis thaliana KAT1 channel (hereafter CPP) dissolved in MilliQ Water. Peptide stock concentration is 15 mM) (Peptide was purchased from CASLO ApS as a lyophilized powder, purity ≥ 98%)
  7. NaCl*, stock concentration 5 M (Sigma-Aldrich, catalog number: 746398 )
  8. HEPES*, stock concentration 1 M, pH 7.4 (Sigma-Aldrich, catalog number: RDD002 )
  9. NaOH*, stock concentration 10 M (Sigma-Aldrich, catalog number: S8045 )
  10. MgCl2*, stock concentration 2 M (Sigma-Aldrich, catalog number: M8266 )
  11. β-mercaptoethanol*, stock concentration 100 mM (Sigma-Aldrich, catalog number: M6250 )
  12. MilliQ Water*
  13. Sample buffer (see Recipes)
*Note: These items can be purchased from any suitable vendor.

Equipment

  1. Pipettors (P10, P20, P200, P1000)*
  2. Microcalorimeter (Malvern Panalytical, model: MicroCal VP-ITC )
  3. Vacuum pump (provided together with MicroCal VP-ITC, Malvern Panalytical, model: ThermoVac, catalog number: 29013182 )
  4. Filling syringe (provided together with MicroCal VP-ITC, Malvern Panalytical)
  5. Plastic tubes, 3 ml (provided together with MicroCal VP-ITC, Malvern Panalytical)
  6. Benchtop refrigerated centrifuge with rotor FA-45-48-1 (Eppendorf, model: 5427 R , catalog number: 5409000210)
  7. Stir bars 7 mm for mixing the solutions within the vacuum pump (ThermoVac) (Malvern Panalytical, catalog number: BAR150020-005 )
  8. UV spectrometer (Eppendorf, BioSpectrometer® basic supplied with Eppendorf μCuvette® G1.0, that is suitable for volumes of 1.5-10 μl, catalog number: 6135000904 )
  9. pH meter*
*Note: These items can be purchased from any suitable vendor.

Software

  1. Origin software (version7, MicroCal, Malvern Instruments Ltd, RRID: SCR_014212)

Procedure

  1. Measurements were carried out at 25 °C using a VP-ITC Microcalorimeter (Malvern Instruments Ltd). The volume of both the reference and the sample cell was 1.4 ml.
  2. The solutions containing the titrant (CPP) and the titrated (14-3-3c) molecules have to be identical to avoid heat changes due to the dilution of the titrant into the sample cell.
    The following table describes the appropriate dilutions required to prepare the solutions containing the titrant (CPP) and the titrated (14-3-3c) molecules at 500 μM and 50 μM concentration respectively:


    As highlighted in the table, since CPP stock is in MilliQ water, in preparing 14-3-3c dilution it is necessary to use the same proportion of MilliQ water used in the preparation of the CPP dilution to have identical solutions for both titrant (CPP) and titrated molecule (14-3-3c). 
  3. Prior to loading MilliQ water and sample solutions into the machine, load them into the 3 ml plastic tubes (see equipment) together with stir bars 7 mm and degas them for 5 min by using the Vacuum pump (ThermoVac). This instrument is indeed designed to allocate the 3 ml plastic tubes and favorite the degassing by combining the vacuum with a gently stirring.
  4. Centrifuge both protein and titrant at 16,000 x g, for 5 min, at 4 °C.
  5. Filling procedure of reference/sample cell (Video 1):
    1. Insert the 2.5 ml glass syringe until you gently touch the bottom of the cell with the tip of the syringe.
    2. Inject slowly and constantly the solution, removing, at the same time, the needle from the cell. In this way, the cell is filled from the bottom to the top, and the operation avoids the formation of bubbles.
    3. Position the tip of the syringe on the ledge present just below the visible portion of the cell port and draw the liquid to remove the excess of liquid from the cell.

    Video 1. Filling procedure of reference/sample cell. The video shows the steps necessary for the proper loading of the solutions into the reference/sample cells (see Steps 5a-5c). For simplicity, the video describes only the filling of the sample cell.

  6. Fill the reference cell with MilliQ water.
  7. Fill the sample cell with the protein (in this case 14-3-3c, 50 μM).
    Note: In order to obtain a reliable measurement, both reference and sample cells must be precisely filled. This operation can be performed by loading a larger volume of solution (2 ml recommended) compared to the maximum capacity of the cells. Often, the low yield of purified protein limits this operation. Nevertheless, use at least 1.6 ml to be sure to appropriately load the sample. The proper loading of the solutions is a critical step in order to avoid the formation of bubbles, which severely impair the measurements.
  8. The volume of the injection syringe is 300 μl. Thus, to appropriately load the titrant, prepare at least 500 μl of the titrant sample.
  9. Load the titrant (in this case CPP, 500 μM) into the injection syringe with the following procedure (Video 2):
    1. Connect the plastic tubing of a filling syringe to the filling port of the injection syringe.
    2. Place the titrant sample in a glass filling tube, and insert it into the pipette stand of the machine.
    3. Slowly withdraw the plunger of the filling syringe to draw up the solution containing the titrant.
    4. Purge the bottom "close fill port" as soon as the titrant solution begins to exit the filling port.
    5. Perform a refill step in order to remove any air bubbles from the injection syringe.

    Video 2. Filling procedure of the injection syringe. The video shows the steps necessary for the proper loading of the titrant into the injection syringe (see Steps 9a-9e).

  10. Titrate CPP using injection volumes of 10 μl. The spacing between consecutive injections is 150 sec. A total of 29 injections are performed.
    Note: The volume of the first injection is 2 μl, 1/5 of the settled injection volume. This is needed to equilibrate the interface between the tip of the injection syringe and the surrounding solution of the sample cell. The heat change caused by this first injection has to be discarded during data analysis. Because of this first 2 μl injection, a total of 292 μl of titrant out of the 300 μl loaded into the syringe will be used and 8 μl will be left into the syringe after the experiment.
  11. It is worth noting that the concentration of the titrant and the titrated molecules, as well as the injection volumes and the spacing time between consecutive injections, is appropriate for the molecules used in this protocol. These parameters must be optimized for every sample.
    Note: In the initial phase of setting parameters, a 10-fold difference in the concentration between the titrant and the titrated molecule should be maintained. This usually ensures a complete saturation of the binding reaction for those interactions having a binding affinity in the nM to μM range. If there is evidence that the binding affinity of the system under study is in the hundred μM to mM range, however, the fold difference of the concentration between the titrant and the titrated molecule should be increased.
      Moreover, the spacing should be set at 180 sec, which is usually a long enough time to ensure the complete recovery of the heat baseline upon titrant injection. In case your measurement requires a different time to return to the baseline, adapt the spacing time to your system. Data extrapolated from measurements performed with different spacing times can be combined, as long as the baseline is completely recovered and the concentration of titrant/titrated molecules and the injection volumes are the same.

Data analysis

Calorimetric data were analysed with Origin software (version7, MicroCal, RRID:SCR_014212) and equations were described for the single-site binding model (Wiseman et al., 1989). It is worth noting that the procedure generally follows the “Instrument Handbook” of MicroCal VP-ITC system (Malvern Instruments Ltd).


Figure 2. ITC measurement of CPP–14-3-3c interaction. A. Raw ITC data of CPP (500 μM) binding to 14-3-3c (50 μM). The area of the negative heat changes (μcal/sec) during successive injections of 10 μl of CPP into the sample cell containing 14-3-3c was calculated by selecting the option “integrate all peaks” from the ITC main control list in order to measure the amount of heat released by each CPP injection of the titration. The area of the peaks is colored in red. B. The quantified heat changes (red filled circles), normalized per mole of CPP injected and plotted against the molar ratio of CPP : 14-3-3c, were fitted to a single-site binding model by selecting the option “One set of sites” in the model fitting list implemented in the software. This generates the fitting curve (blue line) that interpolates the heat changes (red filled circles), and the related values of stoichiometry (N, black labelled), association equilibrium constant (KA, orange labelled), enthalpy (∆H, green labelled), and entropy (∆S black labelled) of the binding reaction. All these values are shown in the blue dotted box. C. Final ITC figure composed by an upper panel (corresponding to panel A) and lower panel (corresponding to panel B). Lower panel shows also the schematic representation of the calculations performed by the software to obtain: ∆H as the difference between the initial H value and the plateau H value (green line with double arrowheads); KA, which is the slope value of the orange line that intercepts the exponential phase of the isothermal curve (blue line); N, which is to the molar ratio (CPP:14-3-3c) at the centre of the isothermal curve (indicated by the black dotted line).

Figure 2 summarizes the processing of the ITC raw data and their subsequent analysis by using a single-binding model needed to obtain the binding curve for the CPP–14-3-3c interaction. Panel A shows negative heat changes (μcal/sec) during successive injections of 10 μl of CPP (500 μM) into the sample cell containing 14-3-3c (50 μM). This behavior, according to the basic ITC principles described in the Background section and illustrated in Figure 1, indicates that the CPP–14-3-3c interaction releases heat, so that the binding reaction is exothermic.

The software automatically integrates the peaks. This operation is graphically represented in Figure 2, Panel A, by the peaks colored in red. A critical aspect in the data analysis is the correct assessment of the integration details: the baseline and the integration range. The software automatically sets the integration details and integrates the peaks. However, there are cases where the signal to noise ratio is low and the automatic calculation of the integration details is not accurate enough. In these cases, both the baseline and the integration range need to be manually adjusted, following the “Instrument Handbook” instructions of MicroCal VP-ITC.

Moreover, the software automatically normalizes the integrated peaks to the CPP concentration, and plots them against the molar ratio of CPP (titrant) : 14-3-3c (titrated molecule) (red filled circles in Figure 2, Panel B). In Panel B, the solid blue line interpolating the red circles represents a nonlinear least-squares fit to a single-site binding model (Wiseman et al., 1989). The software calculates the stoichiometry (N), the association equilibrium constant (KA), the enthalpy (∆H), and the entropy (∆S) of the binding reaction. In Figure 2, Panel B, these values are shown within the blue dotted box. N, KA, and ∆H are directly derived from the isothermal curve, and for this reason they yield a fitting error. On the other hand, ∆S, which is indirectly calculated, does not yield a fitting error.

The Gibbs free energy (∆G) is not calculated by the software, but it can be calculated by the operator (see below).
Figure 2C, which is composed by panels A and B, corresponds to the final ITC figure most commonly used for publication.
For a better comprehension of the operations performed by the software, panel C includes a graphic representation of how the software calculates, starting from the isothermal curve, ∆H, N, and KA.
The binding enthalpy (∆H), which is due to hydrogen bonds and van der Waals interactions, is calculated as the difference between the starting H value and the plateau H value (see green line in Figure 2C). As stated above, the ∆H value is normalized per mole of the titrant.
The stoichiometry (N) corresponds to the molar ratio at the center of the isothermal curve (see the black dotted line in Figure 2C).
Note: An accurate assessment of the concentration of the biomolecules is very important, because the stoichiometry parameter is extremely sensitive to their concentration.
The association equilibrium constant (KA) corresponds to the slope of the line that intercepts the exponential phase of the isothermal curve (see blue line in Figure 2C). It is possible to manually calculate the dissociation equilibrium constant (KD), which is the equilibrium constant more commonly used to describe the affinity of biomolecules, by using the following equation:



The binding entropy (∆S), which is due to the desolvation of biomolecules and eventually to conformational changes induced by the complex formation, is indirectly calculated using the following equation:



The Gibbs free energy (∆G) is indirectly calculated using the following equation:

Notes

  1. As already stated in the "Data analysis" section, the concentration of the biomolecules is extremely important for the calculation of the stoichiometry of the binding relation. Therefore, the first time that the concentration of the biomolecules under study is assessed, the recommendation is to use at least two different methods in order to be sure of the accuracy of their concentration. For example, in this specific case, the first time I quantified the concentration of the 14-3-3c stock, I used two different methods: a Bradford-based method, and a 280 nm absorption-based method using a molar extinction coefficient value (ε) of 14-3-3c, which I theoretically calculated using the ProtParam tool (https://web.expasy.org/protparam/).
  2. The ITC measurements should be repeated at least three times to have enough data for statistics. 
  3. A control measurement should be performed by titrating the titrant against the buffer, in order to evaluate the heat change caused by the titrant injection per se. If the titrant injection causes a significant heat change, this measurement should be used to subtract the heat change originating from the dilution of the titrant into the buffer from the titrant - titrated molecule binding relation.

Recipes

  1. Sample buffer
    100 mM NaCl
    30 mM HEPES (pH 7.4)
    10 mM MgCl2
    2 mM β-mercaptoethanol
    Notes:
    1. Do not use Tris-HCl and DTT because they can severely affect the measurements by producing intense heat changes. If it is necessary to maintain a reductive state, the recommendation is to use β-mercaptoethanol.
    2. The above buffer works with the protein and the molecule used in this specific protocol. Buffer must be optimized for other samples.

Acknowledgments

This protocol was adapted from Saponaro et al. (2017), and generally follows the “Instrument Handbook” of the MicroCal VP-ITC system (Malvern Instruments Ltd). This work was supported by Lincean Academy (Italian Science Academy), Giuseppe Levi foundation.
Conflicts of interest or competing interests: none.

References

  1. Saponaro, A., Porro, A., Chaves-Sanjuan, A., Nardini, M., Rauh, O., Thiel, G. and Moroni, A. (2017). Fusicoccin activates KAT1 channels by stabilizing their interaction with 14-3-3 proteins. Plant Cell 29(10): 2570-2580.
  2. Wiseman, T., Williston, S., Brandts, J. F. and Lin, L. N. (1989). Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179(1): 131-137.

简介

该方案可用于使用等温滴定量热法(ITC,Malvern)分析可溶性蛋白质和靶配体分子之间的直接相互作用。 ITC允许通过提供相互作用的化学计量,平衡结合常数和热力学参数来对无标记,非固定和溶液内生物分子之间的结合进行生物物理表征。 ITC监测由大分子相互作用引起的热变化(释放和/或吸收),而不限制大分子的缓冲液和分子量。

【背景】大分子相互作用是关键的细胞事件,因为它们形成信号转导途径的基础。因此,大分子相互作用是研究的重要领域,因为它们允许更深入地理解作为生理学和病理生理学过程基础的分子机制,以及能够调节引起疾病的大分子结合事件的药物的合理设计。

在这种情况下,等温滴定量热法(ITC)是一种用于表征大分子相互作用的强大技术。 ITC确定两个分子结合时发生的热变化。可以吸收热量(吸热反应)或释放(放热反应)。 ITC通过确定由仪器的加热器提供给参比和样品池的差异功率来监测这种热变化,这是在结合反应期间抵消两个电池之间的任何温差所需的,使得参考之间没有温度差异。和样品细胞(图1)。


图1.等温滴定量热法(ITC)的原理。 A.等温滴定量热仪器的卡通表示,包括:填充MilliQ水的参比池;含有生物分子的样品细胞;和含有另一种结合分子(配体)的自动注射器,用于将配体滴定到样品细胞中。样品和参比细胞被绝热夹套包围。该系统能够检测参考和样品池之间的温差,并通过两个加热器向参比池和样品池供电,以保持它们之间不存在温差(ΔT= 0)。仪器的输出是在参考和样品池之间维持ΔT= 0所需的功率(μcal/ sec)。 B-d。由配体 - 生物分子结合诱导的参照和样品细胞之间的温差被转换成在结合反应期间使两个细胞恢复到相同温度所需的功率。随着滴定的进行,样品池中的生物分子被配体饱和,因此发生较少的相互作用,因此热量变化减少(C),直到生物分子完全饱和,仪器仅检测到由于稀释而导致的热量变化。配体(D)。

ITC提供了关于大分子相互作用性质的重要信息:结合化学计量(N);结合反应的热力学参数(焓,ΔH,熵,ΔS和吉布斯自由能,ΔG);相互作用的强度(平衡缔合常数K A ,可以从中得出更常用的平衡解离常数K D )。

在用于表征大分子相互作用的方法中,ITC具有两个主要优点:i)生物分子可在溶液中自由移动且未标记,这确保了对结合事件的直接表征,通过标记和/或对分子的限制无偏由于它们固定在表面上而引起的运动; ii)ITC是唯一允许通过提供结合亲和力以及其他关键信息即,结合化学计量和热力学参数来详细表征结合事件的方法。即使没有结构数据,这些信息也可以帮助理解结合反应的分子机制。此外,它们可用作补充数据以验证结构结果。

最近,我提出了晶体学和功能数据,显示K + 内向整流器KAT1(K + 拟南芥 1)通道受直接调控结合14-3-3蛋白质(Saponaro et al。,2017)。特别是,我在KAT1的C末端鉴定了一个14-3-3模式III结合位点,并与烟草14-3-3蛋白(14-3-3c)共结晶,以描述原子中的蛋白质复合物。详情。通过ITC测量合成的KAT1 C-末端磷酸肽(CPP)和14-3-3c之间的相互作用来补充/支持结构结果。 ITC用于量化14-3-3c-CPP结合反应的化学计量,平衡结合亲和力和热力学参数。

该协议的目的是提供ITC实验设置过程的详细描述,突出关键步骤和相关问题,同时提供克服此类问题的既定策略。此外,本协议描述了14-3-3c / CPP相互作用中单一结合事件的ITC测量的分析。

关键字:ITC, 量热法, 大分子互作, 结合亲和力, KAT1通道, 14-3-3蛋白质


材料和试剂

  1. 移液器吸头(20μl,200μl和1,000μl)*
  2. 玻璃注射器2.5 ml,18GA,8.5IN,PT3(Malvern Panalytical,目录号:SYN161714)
  3. 一次性硼硅酸盐玻璃管,0.7 ml,6 x 50 mm(O.D。x Height)(DWK Life Sciences,Kimble Chase,目录号:73500-650)
  4. 0.22μm真空过滤器*
  5. 纯化的烟草(Nicotiana tabacum)14-3-3c蛋白在 E中重组表达。大肠杆菌。蛋白质原液浓度为100μM
    注意:如Saponaro等人所述,表达并纯化蛋白质。 (2017)。
  6. 合成的磷酸肽*(对应于溶解于MilliQ水中的拟南芥 KAT1通道(以下称CPP)的最后5个残基。肽储备浓度为15mM)(肽以CASLO ApS购自冻干粉末,纯度≥98%)
  7. NaCl *,储备浓度5 M(Sigma-Aldrich,目录号:746398)
  8. HEPES *,储备浓度1M,pH 7.4(Sigma-Aldrich,目录号:RDD002)
  9. NaOH *,储备浓度10 M(Sigma-Aldrich,目录号:S8045)
  10. MgCl 2 *,储备浓度2 M(Sigma-Aldrich,目录号:M8266)
  11. β-巯基乙醇*,储备浓度100 mM(Sigma-Aldrich,目录号:M6250)
  12. MilliQ Water *
  13. 样品缓冲液(见食谱)
*注意:这些项目可以从任何合适的供应商处购买。

设备

  1. 移液器(P10,P20,P200,P1000)*
  2. 微量热仪(Malvern Panalytical,型号:MicroCal VP-ITC)
  3. 真空泵(与MicroCal VP-ITC一起提供,Malvern Panalytical,型号:ThermoVac,目录号:29013182)
  4. 灌装注射器(与MicroCal VP-ITC,Malvern Panalytical一起提供)
  5. 塑料管,3毫升(与MicroCal VP-ITC,Malvern Panalytical一起提供)
  6. 带转子的台式冷冻离心机FA-45-48-1(Eppendorf,型号:5427 R,目录号:5409000210)
  7. 搅拌棒7 mm,用于混合真空泵(ThermoVac)内的溶液(Malvern Panalytical,目录号:BAR150020-005)
  8. 紫外光谱仪(Eppendorf,BioSpectrometer ® basic与EppendorfμCuvette® G1.0一起提供,适用于1.5-10μl的体积,目录号:6135000904)
  9. pH计*
*注意:这些商品可以从任何合适的供应商处购买。

软件

  1. Origin软件(版本7,MicroCal,Malvern Instruments Ltd,RRID:SCR_014212)

程序

  1. 使用VP-ITC微量热计(Malvern Instruments Ltd)在25℃下进行测量。参比和样品池的体积均为1.4ml。
  2. 含有滴定剂(CPP)和滴定的(14-3-3c)分子的溶液必须相同,以避免由于滴定剂稀释到样品池中而引起的热量变化。
    下表描述了分别以500μM和50μM浓度制备含有滴定剂(CPP)和滴定(14-3-3c)分子的溶液所需的适当稀释度:表


    如表中所示,由于CPP库存在MilliQ水中,在准备14-3-3c稀释时,必须使用相同比例的MilliQ水制备CPP稀释液,以获得相同的滴定剂溶液(CPP) )和滴定分子(14-3-3c)。 
  3. 在将MilliQ水和样品溶液装入机器之前,将它们与7 mm搅拌棒一起装入3 ml塑料管(见设备)中,然后使用真空泵(ThermoVac)将它们脱气5分钟。该仪器确实设计用于分配3毫升塑料管,并通过将真空与温和搅拌相结合而最喜欢脱气。
  4. 将蛋白质和滴定剂在16,000 x g 下离心5分钟,在4°C下离心。
  5. 参考/样品池的填充程序(视频1):
    1. 插入2.5毫升玻璃注射器,直到用注射器尖端轻轻触摸电池底部。
    2. 缓慢注入溶液,同时从细胞中取出针头。以这种方式,电池从底部到顶部填充,并且操作避免了气泡的形成。
    3. 将注射器的尖端定位在细胞端口的可见部分正下方的凸缘上,并抽出液体以从细胞中除去过量的液体。

    视频1.参考/样品池的填充程序。 视频显示了将溶液正确加载到参考/样品池中所需的步骤(参见步骤5a-5c)。为简单起见,视频仅描述样本单元的填充。

  6. 用MilliQ水填充参比池。
  7. 用蛋白质填充样品池(在这种情况下为14-3-3c,50μM)。
    注意:为了获得可靠的测量,必须精确填充参考和样品池。与细胞的最大容量相比,可以通过加载更大体积的溶液(推荐2ml)来进行该操作。通常,纯化蛋白质的低产率限制了该操作。尽管如此,使用至少1.6毫升,以确保适当加载样品。正确加载溶液是一个关键步骤,以避免形成气泡,严重影响测量。
  8. 注射器的体积为300μl。因此,为了适当加载滴定剂,准备至少500μl滴定剂样品。
  9. 按照以下步骤将滴定剂(在本例中为CPP,500μM)装入注射器(视频2):
    1. 将注射器的塑料管连接到注射器的注射口。
    2. 将滴定剂样品放入玻璃填充管中,并将其插入机器的移液器支架中。
    3. 缓慢抽出灌装注射器的柱塞,抽出含有滴定剂的溶液。
    4. 一旦滴定剂溶液开始离开填充口,就清除底部的“密闭填充口”。
    5. 执行重新填充步骤以清除注射器中的任何气泡。

    视频2.注射器的灌装步骤。视频显示了将滴定剂正确装入注射器所需的步骤(参见步骤9a-9e)。

  10. 使用10μl的注射体积滴定CPP。连续注射之间的间隔为150秒。共进行了29次注射。
    注意:第一次注射的体积是2μl,是固定注射量的1/5。这需要平衡注射器的尖端和样品池的周围溶液之间的界面。在数据分析期间必须丢弃由第一次喷射引起的热量变化。由于首次注射2μl,将使用加入注射器的300μl中总共292μl滴定剂,并在实验后将8μl留在注射器中。
  11. 值得注意的是,滴定剂和滴定分子的浓度,以及注射体积和连续注射之间的间隔时间适合于该方案中使用的分子。必须针对每个样品优化这些参数。
    注意:在设定参数的初始阶段,应保持滴定剂和滴定分子之间浓度的10倍差异。这通常确保对于在nM至μM范围内具有结合亲和力的那些相互作用的结合反应的完全饱和。如果有证据表明所研究系统的结合亲和力在100μM至mM范围内,则应增加滴定剂与滴定分子之间浓度的倍数差异。
      此外,间距应设定为180秒,这通常是足够长的时间,以确保在滴定剂注入后完全恢复热基线。如果您的测量需要不同的时间返回基线,请将间隔时间调整到您的系统。只要基线完全恢复并且滴定剂/滴定分子的浓度和注射体积相同,就可以组合从不同间隔时间进行的测量推断的数据。

数据分析

用Origin软件(版本7,MicroCal,RRID:SCR_014212)分析量热数据,并描述了单点结合模型的方程式(Wiseman 等人,,1989)。值得注意的是,该程序通常遵循MicroCal VP-ITC系统(Malvern Instruments Ltd)的“仪器手册”。


图2. CPP-14-3-3c相互作用的ITC测量。 A. CPP(500μM)与14-3-3c(50μM)结合的原始ITC数据。通过从ITC主控制列表中选择“积分所有峰”选项,计算连续注入10μlCPP到含有14-3-3c的样品池中的负热变化面积(μcal/ sec),以便测量每次CPP注射滴定释放的热量。峰的区域用红色着色。 B.量化的热变化(红色圆圈),相对于CPP:14-3-3c的摩尔比标准化的每摩尔注射的CPP标准化,通过选择“一组”选项拟合到单点结合模型。网站“在软件中实现的模型拟合列表中。这将生成拟合曲线(蓝线),插入热变化(红色圆圈),以及化学计量的相关值(N,黑色标记),缔合平衡常数(K A ,橙色标记) ,焓(ΔH,绿色标记)和结合反应的熵(ΔSblack标记)。所有这些值都显示在蓝色虚线框中。 C.由上面板(对应于面板A)和下面板(对应于面板B)组成的最终ITC图。下图还显示了由软件执行的计算的示意图,以获得:ΔH作为初始H值和平台H值之间的差值(带双箭头的绿线); K A ,它是截取等温曲线指数相位的橙色线的斜率值(蓝线); N,即等温曲线中心的摩尔比(CPP:14-3-3c)(用黑色虚线表示)。

图2通过使用获得CPP-14-3-3c相互作用的结合曲线所需的单结合模型,总结了ITC原始数据的处理及其后续分析。图A显示在将10μlCPP(500μM)连续注入含有14-3-3c(50μM)的样品池期间的负热变化(μcal/ sec)。根据背景技术部分和图1中所示的基本ITC原理,这种行为表明CPP-14-3-3c相互作用释放热量,因此结合反应是放热的。

该软件自动集成峰值。该操作在图2的图A中用红色着色的图形表示。数据分析的一个关键方面是对集成细节的正确评估:基线和集成范围。该软件自动设置集成细节并集成峰值。然而,存在信噪比低并且集成细节的自动计算不够准确的情况。在这些情况下,需要根据MicroCal VP-ITC的“仪器手册”说明手动调整基线和积分范围。

此外,软件自动将积分峰标准化为CPP浓度,并将它们与CPP(滴定剂):14-3-3c(滴定分子)的摩尔比进行绘图(图2中的红色圆圈,图B)。在图B中,插入红色圆圈的实线蓝线表示与单点结合模型的非线性最小二乘拟合(Wiseman et al。,1989)。该软件计算结合反应的化学计量(N),缔合平衡常数(K A ),焓(ΔH)和熵(ΔS)。在图2的面板B中,这些值显示在蓝色虚线框内。 N,K A 和ΔH直接来自等温曲线,因此它们产生拟合误差。另一方面,间接计算的ΔS不会产生拟合误差。

吉布斯自由能(ΔG)不是由软件计算的,但可以由操作员计算(见下文)。
图2C由面板A和B组成,对应于最常用于出版的最终ITC图形。
为了更好地理解软件执行的操作,面板C包括软件如何计算的图形表示,从等温曲线开始,ΔH,N和K A 。
由氢键和范德华相互作用引起的结合焓(ΔH)计算为起始H值和平台H值之间的差值(参见图2C中的绿线)。如上所述,ΔH值相对于每摩尔滴定剂标准化。
化学计量(N)对应于等温曲线中心的摩尔比(参见图2C中的黑色虚线)。
注意:准确评估生物分子的浓度非常重要,因为化学计量参数对其浓度非常敏感。
缔合平衡常数(K A )对应于截取等温曲线的指数相的线的斜率(参见图2C中的蓝线)。可以通过使用以下等式手动计算解离平衡常数(K D ),这是更常用于描述生物分子亲和力的平衡常数:



结合熵(ΔS),由于生物分子的去溶剂化和最终由复合物形成诱导的构象变化,使用以下等式间接计算:



吉布斯自由能(ΔG)使用以下等式间接计算:

笔记

  1. 1.如“数据分析”部分中所述,生物分子的浓度对于计算结合关系的化学计量非常重要。因此,第一次评估所研究的生物分子的浓度时,建议使用至少两种不同的方法以确保其浓度的准确性。例如,在这种特殊情况下,我第一次量化了14-3-3c原料的浓度,我使用了两种不同的方法:基于Bradford的方法,以及使用摩尔消光系数值的280 nm吸收法14-3-3c的(ε),我在理论上使用ProtParam工具计算出来( https://web.expasy .ORG /的protParam / )。
  2. ITC测量应至少重复三次,以获得足够的统计数据。 
  3. 应通过滴定剂与缓冲液滴定来进行对照测量,以评估滴定剂注射本身引起的热量变化。如果滴定剂注入导致显着的热量变化,则应使用此测量值来减去由滴定剂 - 滴定分子结合关系将滴定剂稀释到缓冲液中产生的热量变化。

食谱

  1. 样品缓冲液
    100 mM NaCl
    30 mM HEPES(pH 7.4)
    10mM MgCl 2
    2mMβ-巯基乙醇
    注意:
    1. 不要使用Tris-HCl和DTT,因为它们会通过产生强烈的热量变化严重影响测量。如果有必要保持还原状态,建议使用β-巯基乙醇。
    2. 上述缓冲液与该特定方案中使用的蛋白质和分子一起使用。必须针对其他样本优化缓冲区。

致谢

该方案改编自Saponaro 等人(2017),并且通常遵循MicroCal VP-ITC系统(Malvern Instruments Ltd)的“仪器手册”。这项工作得到了Lincean Academy(意大利科学院),Giuseppe Levi基金会的支持。
利益冲突或利益冲突:无。

参考

  1. Saponaro,A.,Porro,A.,Chaves-Sanjuan,A.,Nardini,M.,Rauh,O.,Thiel,G。和Moroni,A。(2017)。 Fusicoccin通过稳定与14-3-3蛋白的相互作用来激活KAT1通道。 em>植物细胞 29(10):2570-2580。
  2. Wiseman,T.,Williston,S.,Brandts,J.F。和Lin,L。N.(1989)。 使用新的滴定热量计快速测量结合常数和结合热。 Anal Biochem 179(1):131-137。

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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Saponaro, A. (2018). Isothermal Titration Calorimetry: A Biophysical Method to Characterize the Interaction between Label-free Biomolecules in Solution. Bio-protocol 8(15): e2957. DOI: 10.21769/BioProtoc.2957.
  2. Saponaro, A., Porro, A., Chaves-Sanjuan, A., Nardini, M., Rauh, O., Thiel, G. and Moroni, A. (2017). Fusicoccin activates KAT1 channels by stabilizing their interaction with 14-3-3 proteins. Plant Cell 29(10): 2570-2580.
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