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JoVE Journal Medicine

Medicine

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

Published: March 1, 2024 doi: 10.3791/66610
*1, *2, *2, 1, 2
1Department of Tibetan Medicine, University of Tibetan Medicine, 2Innovative Institute of Chinese Medicine and Pharmacy / Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine
* These authors contributed equally

Abstract

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

Introduction

Vicatia thibetica H. Boissieu, a plant in the umbelliferous family, is commonly used in Tibetan medicine and represents one of the essential components of the five basic ingredients (Polygonatum sibiricum Delar. ex Redoute, Asparagus cochinchinensis (Lour.) Merr, Vicatia thibetica H. Boissieu, Oxybaphus himalaicus Edgew., and Gymnadenia conopsea (L.) R. Br.)1. Mainly distributed in southwest China, such as northwest Yunnan, western Sichuan, Tibet, and other areas, its dried root serves as a local substitute for Angelica1. The root, known for its fragrance, is often utilized as a stew seasoning, and the leaves, referred to as Tibetan celery, contribute to delectable dishes. Thus, Vicatia thibetica holds significance not only as a unique medicinal plant but also as a food source in Tibet.

Both domestic and international research indicates that Vicatia thibetica possesses blood-replenishing and qi (power or energy)-invigorating properties, beneficial for regulating menstruation. It is employed to address symptoms of irregular menstrual dysmenorrhea caused by palpitation and blood deficiency, exhibiting pharmacological effects such as antioxidant regulation of body immunity2. The alcohol extract of Vicatia thibetica has demonstrated the ability to restore body mass and organ index in immunocompromised mice induced by cyclophosphamide. Additionally, it increases the activity of superoxide dismutase in serum and reduces the content of malondialdehyde, suggesting an improvement in antioxidant capacity and a balancing effect on lipid peroxidation2,3. Simultaneously, it enhances the number of blood cells and hemoglobin, which not only objectively reflects the body's hematopoietic function but also plays a crucial role in the immune system2,3.

Umbelliferone, characterized by acicular crystals and a bitter taste, possesses a small molecular weight, is volatile, and can be distilled with steam. It sublimates easily, has low solubility in water, and high solubility in organic matter. As one of the main chemical components of Vicatia thibetica, umbelliferone exhibits immunomodulatory effects on cellular immunity, humoral immunity, and non-specific immunity in hydrocortisone-induced immunosuppression mouse models4,5.

The cell surface molecule CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed in immune cells6. Its homologous ligand, CD154, also known as CD40L, is a type II transmembrane protein expressed by activated T lymphocytes. CD40 activation has the capacity to up-regulate the expression of co-stimulatory molecules on the surface of dendritic cells (DC) and monocytes. This process promotes the antigen presentation function of major histocompatibility complex (MHC) molecules and further activates CD8+ T cells7.

Macrophages play a crucial role in the formation and regulation of the tumor microenvironment, and CD40 activation can enhance the remodeling of the tumor microenvironment by macrophages8. CD40 signal activation significantly influences the proliferation and activation of B cells. B cells, when activated by CD40, can function as effective antigen-presenting cells. They present antigens, generate effector T cell activity, and thereby contribute to anti-tumor effects9. Moreover, CD40 activation in tumor cells can induce apoptosis and inhibit tumor growth10. CD40 primarily transduces signals by controlling the activity of non-receptor tyrosine protein kinases, including Lyn, Fyn, Syk, and others. Additionally, it has the ability to stimulate Bcl-xL, Cdk4, and Cdk6 proteins, activate Rel/NF-kB transcription factors, and phosphorylate CG-2 and PI3K10.

The Differential Scanning Fluorescence (DSF) method is widely employed to assess the impact of various environmental conditions, such as buffer composition, temperature, and small molecule ligands, on the thermal stability of protein structures. The commonly utilized dye for DSF is an orange, environmentally sensitive, hydrophobic dye. Under normal conditions, the protein structure is folded, concealing its hydrophobic segment internally. As the temperature increases, the protein's hydrophobic region becomes more exposed, resulting in a gradual breakdown of the natural protein structure. The dye selectively binds to this exposed protein portion, amplifying its fluorescence. Tm values are then calculated by monitoring changes in the fluorescence signal detection11. To a certain extent, variations in Tm values can gauge shifts in protein stability due to mutations, changes in buffers, or ligand binding. Furthermore, it can indicate structural alterations during the protein folding process12. This approach offers precise data, a broad temperature range, high sensitivity, and minimal protein sample loss13.

In this study, fluorescence determination was conducted using a fluorescent microplate reader instead of a fluorescence quantitative polymerase chain reaction (PCR) apparatus. This modification enables DSF detection in laboratories lacking a fluorescent quantitative PCR instrument, making the method less complex and reducing the steps required for instrument setup, thereby simplifying the experimental process. However, there are certain drawbacks to this approach. While the complexity is reduced, the procedure becomes more cumbersome. Manual fluorescence detection at different temperatures is necessary, and automatic and continuous collection of fluorescence from the system cannot be achieved. Thus, this study utilized the DSF technique to explore the interaction between CD40 protein and umbelliferone, providing novel insights into the molecular mechanisms of Tibetan medicine.

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Protocol

The compound solution, protein solution, and dye were introduced into a PBS solution. Subsequently, the samples underwent gradual heating using a digital heating shaking dry bath, and the thermal stability of the proteins was evaluated by measuring the change in fluorescence intensity within the complex system. The detailed steps are outlined below, and Figure 1 illustrates an overview of the protocol.

1. Solution preparation

  1. Prepare a 1 mM umbelliferone solution by adding 0.1 mg of umbelliferone to 616.75 µL of DMSO solution (see Table of Materials).
  2. Prepare a 200 µg/mL CD40 protein solution by adding 0.1 mg of CD40 to 500 µL of ddH2O solution (see Table of Materials).
  3. Prepare a 500x orange dye solution by adding 1 µL of 5000x orange dye to 9 µL of phosphate-buffered saline (PBS) (see Table of Materials).
    NOTE: Perform the above operations on ice, and since the pipette has a minimum range of 0.1 µL, initially dilute the orange protein-dye by a factor of 10 to facilitate subsequent use.

2. Dye performance testing

  1. Add the 500x orange dye to a PBS solution and dilute it to achieve dye: PBS ratios of 1:500, 1:1000, 1:2000, and 1:4000.
    NOTE: Ensure that the final concentration of DMSO does not exceed 2% (v/v).
  2. Turn on the fluorescent microplate reader and preheat it for 10 min.
  3. Open the computer and the data acquisition software in sequence (see Table of Materials).
  4. Click on Instrument, choose the corresponding fluorescence microplate model, and select OK in the data acquisition software.
    NOTE: Wait for the fluorescence microplate to finish preheating and display the temperature on the panel before opening the data acquisition software to ensure a successful connection between the instrument and the software.
  5. Click on Acquisition Settings and select Fluorescence in the data acquisition software.
  6. Edit the program in the Wavelengths interface: Lm1 = 470 nm, 570 nm, and then select OK.
    NOTE: The orange dye can be excited with UV light at 300 nm or visible light at 470 nm, and emission can be measured at 570 nm.
  7. Add different concentrations of orange dye (1:500, 1:1000, 1:2000, and 1:4000) and PBS to 96-well plates at 100 µL/well.
    NOTE: Dissolve the solution in DMSO before adding it to PBS, and vortex it at 2000 x g thoroughly during the preparation process to ensure complete dissolution due to the dye's tendency to precipitate.
  8. Place the 96-well plate into the detection stage of the instrument, click on the Read button in the data acquisition software, and measure the fluorescence of each dye concentration 13 times at room temperature, with an interval of 2 min each time.
  9. Click on the Export button after each determination, select Export to XML XLS TXT, choose All Plates, then select Plate in Output Format, and finally click on OK to save the data in a folder in "XLS" format for further analysis.
    NOTE: These steps help determine the impact of continuous measurement on the autofluorescence of the orange dye at room temperature and identify the optimal staining concentration.
  10. Turn on the digital heating shaking dry bath, set the heating temperature to 35 °C, and the heating time to 2 min.
  11. Prepare the optimal dilution ratio of dye in a 1.5 mL microcentrifuge tube and place it in the digital heating shaking dry bath for 2 min.
  12. Add PBS solution and the staining solution, heated at 35 °C, to the 96-well plate at a volume of 100 µL per well.
  13. Place the 96-well plate into the detection stage of the instrument, and click on the Read button in the data acquisition software.
  14. Set the heating temperature to 40 °C and the heating time to 2 min.
  15. Pipette the dye solution in the 96-well plate back into the 1.5 mL microcentrifuge tube and heat it in the instrument for 2 min.
  16. Repeat the operations of steps 2.12-2.15 and step 2.9 to test the absorbance of the dye solution at 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, and 95 °C, respectively.
    NOTE: These steps help determine the stability of the autofluorescence of the orange dye under continuous heating in a gradient from 35 °C to 95 °C. It is crucial to work swiftly to avoid rapid temperature drops or adding the wrong liquid, which could lead to experimental errors.

3. Detection of the temperature melting (Tm) of the protein

  1. Combine CD40 protein and orange dye with PBS solution to achieve final concentrations of 5 µg/mL, 10 µg/mL, 15 µg/mL, 20 µg/mL, and 1:500, respectively.
  2. Repeat the operations outlined in steps 2.2-2.6 and steps 2.10-2.16 to assess the absorbance of the CD40 protein solution at temperatures of 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, and 95 °C, respectively.
  3. Reiterate step 2.9 to save and analyze the data, and subsequently calculate the Tm value using data analysis software (see Table of Materials).
  4. Add CD40 protein, umbelliferone, and orange dye to the PBS solution to obtain final concentrations of 5 µg/mL, 10 µg/mL, 15 µg/mL, 20 µg/mL, 10 µM, and 1:500, respectively.
    NOTE: Vigorous vortexing during solution preparation is essential to ensure even distribution of the dye, CD40 protein, and umbelliferone in PBS.
  5. Repeat the operations of steps 3.2-3.3 to measure the absorbance of the CD40-umbelliferone complexes solution at temperatures of 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, and 95 °C, respectively.
    NOTE: These steps were conducted to determine the Tm values of CD40 protein and CD40-umbelliferone complexes, aiming to identify the optimal CD40 protein binding concentration to umbelliferone.

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Representative Results

The orange dye consistently exhibited stable fluorescence excitation at Ex = 470 nm and Em = 570 nm, both at room temperature and elevated temperatures. An optimal dilution ratio of 1:500 was determined (Figure 2A,B). Detection of the Tm value proved challenging when the concentration of CD40 protein was below 15 µg/mL (Figure 3A,B). However, at a concentration of 15 µg/mL, a stable Tm value of 51.82 °C was detectable, which decreased to 45.79 °C with an increase in CD40 protein concentration (Figure 3C,D).

As depicted in Figure 3, it is evident that CD40 protein binding to umbelliferone influences the Tm value of CD40 protein. Specifically, Tm values of CD40 protein at concentrations of 15 µg/mL and 20 µg/mL, as well as 10 µM umbelliferone, increased to 62.08 °C and 65.27 °C, respectively.

Figure 1
Figure 1: Overview of the protocol. Flowchart of the process of recording the binding capacity of CD40 protein to umbelliferone based on the DSF principle using a fluorescent microplate reader. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Screening of orange dye dilution ratios and stability tests. (A) Fluorescence stability of dyes with different dilution ratios at room temperature. (B) Fluorescence stability of dyes with a dilution ratio of 1:500 at 35-95 °C. The error bars denote mean ± SEM, n = 4. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Result plots of using a fluorescent microplate reader to record the binding capacity of CD40 protein to umbelliferone according to the DSF principle. (A-D) Tm Profiles of 5 µg/mL, 10 µg/mL, 15 µg/mL, 20 µg/mL CD40 Protein and CD40 + 10 µM umbelliferone, respectively. The error bars denote mean ± SEM, n = 4. Please click here to view a larger version of this figure.

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Discussion

DSF, also known as the thermal shift assay or thermal fluorescence assay, is a technique employed to detect the process of thermal denaturation of proteins in samples by monitoring changes in the fluorescence signal of the test sample or dye during a slow, programmed temperature increase. Initially established by Pantoliano14, DSF serves as a high-throughput method. The main procedure involves elevating the temperature on a computer-controlled heated plate, emitting excitation light using a long-wavelength ultraviolet lamp, and capturing changes in fluorescent signal intensity resulting from the binding of fluorescent dyes to proteins through a charge-coupled device phase-forming camera.

The Tm value, a crucial indicator characterizing the higher-level structure of proteins, represents the temperature corresponding to half of the denaturation of the protein drug during a temperature increase. Higher Tm values suggest a greater temperature requirement for protein denaturation, indicating a more stable protein structure. The currently recognized method for detecting Tm values is differential scanning calorimetry (DSC), which was initiated in the 1960s15. DSC measures the difference in power delivered to the object being tested and a reference object as a function of temperature under programmed temperature control to obtain the amount of heat absorbed and discharged. It stands as the gold standard for thermal stability analysis16,17.

DSF has gained widespread use due to three advantages over DSC: (1) Less sample consumption: DSC typically requires a sample volume of 400 µL, while DSF requires only 20-25 µL. (2) Larger concentration range: DSC generally requires protein concentrations of 0.2-2 mg/mL to obtain a significant denaturation peak, whereas DSF can analyze samples in the range of 0.005-200 mg/mL. (3) Higher sample throughput: DSC can analyze only one sample at a time, while DSF can simultaneously scan up to 96 or more samples18. Due to these advantages, DSF is widely utilized in the study of protein stability, protein structure and conformation, protein-ligand interactions, and the effects of protein stabilizers, inhibitors, cofactors, and more.

In addition to DSF and DSC, several other molecular interaction techniques exist. Surface plasmon resonance (SPR) is an optical phenomenon occurring at the interface of two media and can be induced by photons or electrons. It involves total reflection when light is emitted from an optically dense medium into an optically sparse medium. The angle at which surface plasmon resonance occurs is known as the SPR angle. SPR biosensors utilize changes in SPR angles to monitor intermolecular interactions in real-time19,20. MicroScale thermophoresis (MST) is a technique for analyzing biomolecular interactions by detecting changes in the size, charge, and hydration layer of biomolecules caused by binding. It records fluorescence changes in the region irradiated by an infrared laser inside the sample before, during, and after the laser is turned on, allowing for relatively rapid determinations21. Bio-layer interferometry (BLI) measures sensor surface reactions by tracking displacement changes in the interferometry spectrum. In BLI experiments, one molecule is immobilized on the surface of a submersible sensor to detect another molecule that can bind to it. The binding interaction induces interference displacement, and real-time monitoring of this displacement change yields the binding curve22. Isothermal titration calorimetry (ITC) quantifies biomolecular interactions by directly measuring the heat released or absorbed during biomolecular binding. It provides comprehensive thermodynamic information about molecular interactions23. Compared to these molecular interaction technologies, DSF has lower requirements for instruments, consumables, and operating techniques. It can efficiently detect interactions between compounds and proteins in most laboratories.

The operational procedure of DSF technology is relatively straightforward. Initially, white matter samples, reaction buffers, fluorescent dyes, and candidate ligands are combined into a reaction system. This system is then transferred to a fluorescence PCR instrument where heating and detection conditions for fluorescence determination are set up24,25. The most commonly used protein-dye for DSF is currently orange dye, and the detection instrument is typically a fluorescence quantitative PCR instrument26. However, due to the excitation wavelength of orange dye being 470 nm and the emission wavelength being 570 nm, not all fluorescence quantitative PCR instruments on the market are a perfect match. Therefore, this study adopts a fluorescent microplate reader as a new detection instrument to explore protein-ligand interactions.

This approach offers two main advantages over DSF using a fluorescent quantitative PCR instrument. Firstly, the wavelength range of the fluorescent enzyme marker can be customized, making it more compatible with the dye's wavelength range and ensuring more accurate fluorescence detection. Secondly, the program of a fluorescence quantitative PCR instrument cannot be stopped once initiated, making it challenging to troubleshoot any problems during the heating process. In contrast, a fluorescent microplate reader allows for the elimination of interference by iteratively modifying the system ratio and detection conditions throughout the detection process. However, the use of a fluorescent microplate reader for DSF also comes with certain disadvantages, notably the relatively large amount of sample required, typically ranging from 50-200 µL, while traditional DSF typically utilizes 20-25 µL of sample.

In this article, the validation of DSF based on a fluorescent microplate reader was conducted for detecting Tm using CD40 protein and umbelliferone within the concentration range of 5-20 µg/mL. The results demonstrated the method's high specificity and accuracy, affirming its suitability for Tm detection. Subsequent comparison of Tm detection results at different sample concentrations revealed a decreasing trend in the Tm value of CD40 protein as the concentration increased, suggesting a potential concentration effect on Tm detection (Figure 3). Furthermore, the study detected an increase in the Tm value of CD40 protein after the addition of umbelliferone to the system compared to before the addition. This indicates that umbelliferone possesses the ability to bind to CD40 protein, leading to an enhancement in the thermal stability of CD40 protein upon their combination (Figure 3).

The critical steps in the protocol were identified as steps 2.7 to 2.16, the successful completion of which directly influences the subsequent detection of protein fluorescence. In summary, this study established a novel DSF detection method based on DSF technology principles, utilizing CD40 protein and umbelliferone as an example. The method effectively analyzed the binding force between proteins and ligands using a fluorescent microplate reader. Furthermore, this technique can be extended for the analysis of protein stability and research into protein structure, deepening the application of DSF technology in studying cell signaling pathways and exploring disease pathogenesis.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

We express our sincere appreciation for the financial support received from the Connotation Construction Project of the 14th Five-Year Plan of the University of Tibetan Medicine (2022ZYYGH12), the 2022 Open Subjects of the Key Laboratory of Tibetan Medicine and Basic Education of the Ministry of Education at the University of Tibetan Medicine (ZYYJC-22-04), the Key Research and Development Program of Ningxia (2023BEG02012), and the Xinglin Scholar Research Promotion Project of Chengdu University of Traditional Chinese Medicine (XKTD2022013).

Materials

Name Company Catalog Number Comments
CD40 protein MedChemExpress HY-P75408
DMSO Boster Biological Technology Co., Ltd PYG0040
FlexStation 3 multifunctional microplate reader Shanghai Meigu Molecular Instruments Co., LTD FlexStation 3
OriginPro 8 software OriginLab Corporation v8.0724(B724)
Phosphate buffered saline (1x) Gibco 8120485
SoftMax Pro 7.1 Shanghai Meigu Molecular Instruments Co., LTD SoftMax Pro 7.1
SSYPRO orange dye Sigma S5942
Umbelliferone Shanghai Yuanye Biotechnology Co. B21854

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Cairang, N., Zhang, Y., Jiang, H.,… More

Cairang, N., Zhang, Y., Jiang, H., Sonan, T., Wang, X. Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence. J. Vis. Exp. (205), e66610, doi:10.3791/66610 (2024).

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