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 ut....

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.......

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