Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

Summary

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

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 peroxidation^2,3. 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 system^2,3.

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 models^4,5.

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.

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.

Results

The orange dye consistently exhibited stable fluorescence excitation at E_x = 470 nm and E_m = 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 detec...

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-w...

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