As an efficient optical “molecular ruler”, fluorescence resonance energy transfer (FRET) has a wide range of applications in biological macromolecular interactions, immunoassays, nucleic acid detection, etc. In the field of molecular biology, this technique can be used to study protein-protein interactions under physiological conditions in living cells.
Protein-protein interactions play an important role in the whole process of cell life. Due to the extremely complex components in cells, some traditional methods for studying protein-protein interactions, such as yeast two-hybrid and immunoprecipitation, may lose a certain amount of protein. These important information cannot correctly reflect the dynamic changes of protein-protein interactions under the physiological conditions of living cells at that time. FRET technology is a recently developed new technology that facilitates real-time dynamic studies of protein-protein interactions under physiological conditions in living cells.
I. The basic principle of FRET technology
Fluorescence resonance energy transfer means that when two fluorescent chromophores are close enough, when the donor molecule absorbs photons of a certain frequency, it is excited to a higher electronic energy state, and before the electron returns to the ground state, it passes through the dipole. The interaction achieves energy transfer to adjacent acceptor molecules (ie, energy resonance transfer occurs).
FRET is a non-radiative energy transition. Through the electric dipole interaction between molecules, the energy of the excited state of the donor is transferred to the excited state of the acceptor, so that the fluorescence intensity of the donor is reduced, and the acceptor can emit stronger than itself. The characteristic fluorescence (sensitized fluorescence), or no fluorescence (fluorescence quenching), is accompanied by a corresponding shortening or prolongation of the fluorescence lifetime. The efficiency of energy transfer is related to the overlapping degree of the emission spectrum of the donor and the absorption spectrum of the acceptor, the relative orientation of the transition dipoles of the donor and the acceptor, and the distance between the donor and the acceptor. As a resonance energy transfer donor and acceptor pair, fluorescent substances must meet the following conditions:
① The excitation light of the acceptor and the donor should be sufficiently separated;
② The emission spectrum of the donor and the excitation spectrum of the acceptor should overlap.
People have used the organism’s own fluorescence or labeled organic fluorescent dyes on the research objects, and have been successfully used in nucleic acid detection, protein structure, function analysis, immune analysis and organelle structure and function detection and many other aspects. (The absorption spectrum of traditional organic fluorescent dyes is narrow, and the emission spectrum is often accompanied by tailing, which will affect the overlap of the emission spectrum of the donor and the absorption spectrum of the acceptor, and the emission spectra of the donor and acceptor will interfere with each other. Some of the latest reports will luminescence Quantum dots are used in resonance energy transfer studies to overcome the inadequacies of organic fluorescent dyes.
Compared with traditional organic fluorescent dye molecules, the emission spectrum of quantum dots is very narrow and has no tailing, which reduces the overlap of the emission spectrum of the donor and the acceptor and avoids mutual interference; due to the wide spectral excitation range of quantum dots, When it acts as an energy donor, the excitation wavelength can be selected more freely, and the direct excitation of the energy acceptor can be avoided to the greatest extent; by changing the composition or size of the quantum dot, it can emit light of any wavelength in the visible light region, That is to say, it can act as an energy donor for any chromophore whose absorption spectrum is in the visible region, and ensures a good overlap between the emission wavelength of the donor and the absorption wavelength of the acceptor, and increases the resonance energy transfer efficiency. )
Taking two mutants of GFP, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) as examples, the principle is briefly explained: the emission spectrum of CFP and the absorption spectrum of YFP are quite overlapped. Excited by the absorption wavelength, the chromophore of CFP will resonantly transfer energy to the chromophore of YFP with high efficiency, so the emission fluorescence of CFP will weaken or disappear, and the main emission will be the fluorescence of YFP. The energy conversion efficiency between two chromophores is inversely proportional to the 6th power of the spatial distance between them, and is very sensitive to the change of spatial position. For example, to study the interaction between two proteins a and b, a fusion protein can be constructed according to the principle of FRET. This fusion protein consists of three parts: CFP (cyan fluorescent protein), protein b, and YFP (yellow fluorescent protein).
The CFP absorption wavelength of 433 nm was used as the excitation wavelength, and the experiment was dexterously designed so that when the proteins a and b did not interact, CFP and YFP were far apart and could not undergo fluorescence resonance energy transfer. Therefore, the emission wavelength of CFP was detected at 476 nm. Fluorescence; however, when protein a and b interact, the conformational change of protein b due to the action of protein a causes CFP and YFP to be sufficiently close to undergo fluorescence resonance energy transfer. At this time, the emission wavelength of YFP is detected at 527 nm. Fluorescence. The gene encoding the fusion protein is expressed in cells by transgenic technology, so that the protein-protein interaction can be studied under the physiological conditions of living cells.
II. the application of FRET technology
With the deepening of life science research, the research on the mechanism of various life phenomena, especially the protein-protein interaction in cells, has become particularly important. In order to achieve major breakthroughs in research in these areas, technological progress is essential.
The continuous development of some traditional research methods provides extremely favorable conditions for the study of protein-protein interactions, but at the same time, these research methods also have many defects: such as yeast two-hybrid, phosphorylated antibodies, immunofluorescence, radiolabeling, etc. The premise of the application of the method is to break cells or cause damage to cells, and it is impossible to conduct dynamic research on intracellular protein-protein interactions in real time under the physiological conditions of living cells. The application of FRET technology combined with genetic engineering and other technologies just makes up for this defect. The following is the specific application of FRET technology in related life science fields.
1. Detection of changes in enzyme activity
(1) Detection of protein kinase activity in living cells
Protein phosphorylation is an important marker in the process of cell signal transduction, and the study of its enzymatic activity is an important aspect of the study of signal pathways. In the past, the method of measuring enzyme activity mainly used radioactivity and immunochemiluminescence, but the premise was to disrupt the cells and use cell extracts to measure the enzyme activity.
The FRET method can solve this problem very well: for example, Zhang et al. used the FRET principle to design a new probe (a fusion protein): the new probe contains a substrate specific for known protein kinases domain, a phosphorylation recognition domain that binds to the phosphorylated substrate domain. The two ends of this probe protein are the derivatives of GFP, CFP and YFP, and work using the principle of FRET.
When the substrate domain is phosphorylated, the internal folding caused by the binding of the phosphorylation recognition domain to it occurs inside the molecule, and the energy transfer occurs when the two fluorescent proteins approach each other. If the phosphatase acts to dephosphorylate it, the molecule changes reversibly. The research team used several sets of chimeras to study the activity of four known protein kinases: PKA (protein kinase A), Src, Abl, and EGFR (epidermal growth factor receptor).
They transferred the constructed reporter probes into cells and detected changes in kinase activity based on FRET. Following growth factor treatment of cells, several tyrosine kinases were activated within minutes, with a 25%-35% change in activity detected. Activation of PKA with forskolin enhances FRET changes by 25%-50%, and the kinase is activated throughout the cytoplasm. If the reporter probe was localized in the nucleus with the addition of a nuclear localization signal, the FRET changes were greatly delayed, also illustrating the regionality of PKA action. It can be seen that the use of FRET method can well observe the changes of enzyme activity in living cells, and can achieve timing, quantification and localization, which is a very effective research method.
(2) Research on apoptosis
The process of apoptosis can be roughly divided into three different stages: the initial stage – the cell receives a variety of apoptosis-related signals through different pathways; the integration stage – where a variety of signals are integrated and the cell makes a decision to live or die; Execution Period – Once the decision to die is made, an irreversible procedure is about to be entered. Aspartate-specific cysteine protease (cysteinyl aspartate-specific protease, Caspase) plays a key role in the execution phase of apoptosis, and its research has become a hot spot in the field of apoptosis in recent years. The emergence of FRET technology has provided more favorable conditions for this research: Reiko Onuki et al. used FRET technology to study the interaction between Caspase8 and Bid protein. After activation of Caspase8, it acts on Bid protein to split it into two fragments , and then the carboxyl fragment is transferred to mitochondria to release cytochrome c to induce apoptosis.
The researchers fused the two ends of the Bid protein with CFP and YFP respectively, and carefully designed it to allow FRET to occur just before it was cleaved. When the Bid protein was cleaved, the FRET effect disappeared naturally. Therefore, it is a good method to detect the enzymatic activity of Caspase8, and when the Bid protein is fused with CFP and YFP, it can still function normally. Fluorescence can clearly observe its localization in cells. In addition, Markus Rehm and Kiwamu Takemoto et al. used FRET technology to design a fusion reporter protein that can reflect the change of Caspase3 enzyme activity. This reporter protein confirmed that the change of Caspase3 enzyme activity during apoptosis is a very rapid process.
2. Research on membrane proteins
(1) Lateral diffusion of receptor activation effects on the cell membrane
Membrane protein research has always been the focus and difficulty in signaling pathway research. When the cell membrane is locally stimulated by the outside world, the corresponding receptors are activated and then transmit signals into the cell, but is there a lateral effect on the cell membrane before this? Recently, Peter et al. reported in Science that after local stimulation of the cell membrane, the activation effect of membrane receptors can rapidly spread to the entire cell membrane. They fused the membrane receptor EGFR (epidermal growth factor receptor) with GFP, labeled the anti-activated EGFR antibody with Cy3 dye, and labeled the stimulatory factor EGF (epidermal growth factor) with Cy5 dye, so that EGF can be clearly seen in the cell membrane local distribution on . When EGF acts on cells, EGFR activates and binds to its antibody, so GFP and Cy3 dye are sufficiently close to generate FRET. Using this method, it can be clearly observed that after the cell membrane is locally stimulated, the receptor activation effect spreads rapidly to the entire cell membrane.
(2) Localization modification of membrane proteins
We know that membrane proteins are localized in different subregions of the cell membrane, such as lipid rafts and caveolaes, which contain abundant cholesterol, sphingomyelin and signaling proteins. So how do these proteins get to their destinations? Zacharias et al. report in Science that acylation is sufficient to localize these proteins to lipid rafts. Their research was carried out by FRET (fluorescence resonance energy transfer) technology using GFP mutants CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein). Because these proteins do not have intracellular localization sequences, the researchers added various acylation-sensitive sequences to these proteins to study their distribution on the cell membrane. Because the distributed microdomains are very small, FRET can be used to observe when CFP and YFP are co-distributed in the same microdomain. The researchers initially added the acylation sequence of the kinase Lyn to these fluorescent proteins, linking the myristoyl and palmitoyl side chains to the amino termini of CFP and YFP. It was found that the generated FRET signal was very strong, and MCD (5-methyl-β-cyclodextrin), which can remove cholesterol and make the pits and lipid rafts disappear, could not make the fluorescence disappear, so this shows that the fluorescent protein has been very firmly bound. together. When the researchers then replaced the hydrophobic groups on the fluorescent protein with charged groups, they found that aggregate formation was inhibited.
3. Interactions between cell membrane receptors
The signal transmission from external stimuli to cells is generally believed to be through its receptors on the cell membrane. When the ligand binds to the receptor, it causes the receptor conformational change or chemical modification to mediate the signal transmission. However, recent studies on Fas and its homologue TNFR (both trimeric receptors on the cell membrane) have found that they can both spontaneously assemble in the absence of ligands and mediate signal transmission, triggering apoptosis. apoptotic cell apoptosis. Among them, the FRET technology was used in the experiment to identify the trimerization of Fas: Fas was fused with CFP and YFP, respectively. Using this technology, it was convenient to observe whether the Fas monomer was polymerized. Yogesh Patel et al. studied two transmitters dopamine and somatostatin. They found that SSTR5 (the type 5 somatostatin receptor) and D2R (type 2 dopamine receptor) were co-distributed in some neurons in the rat brain, they co-expressed the two, and found that adding a dopaminergic activator could enhance the interaction between SSTR5 and somatostatin Affinity, SSTR5 signaling was inhibited by addition of a dopamine antagonist, and D2R expression restored the coupling of SSTR5 mutants to adenosyl cyclase. This can’t help but think: Is there some kind of connection between the two receptors? Finally, a direct interaction between the two was discovered using the FRET technique (SSTR5 was labeled with a red dye and D2R was labeled with a green dye). Moreover, FRET occurs only when the ligands of both receptors are present, indicating that the interaction between the two receptors occurs when the two receptors are activated.
4. Interactions between intracellular molecules
The small G proteins of the Rho family regulate important physiological functions by regulating actin multimerization. Like other signaling molecules, the effects of these GTPases are very concentrated in time and space, so how to detect the spatiotemporal dynamics of their activity? Woolen cloth? Klaus Hahn et al. reported in Science that a new technology FLAIR (fluorescence activation indicator for Rho proteins) can solve this problem very well: they labeled the domain PDB of PAK1, which can bind and activate Rac-GTP, with the fluorescent dye Alexa, Microinjection into cells expressing GFP and Rac fusion proteins. In this way, when Rac interacts with the PDB, GFP and Alexa are close enough to cause FRET to occur. This method enables real-time detection of the relationship between changes in Rac localization and Rac activation in a living cell.
Matsuda et al. reported in Nature on the activation of Ras and Rap1 in cells, also using FRET technology: they fused the Ras-binding domains (Raf RBD) of Ras and Raf with GFP mutants YFP and CFP. They fused Ras with YFP and RafRBD with CFP. When the two molecules are close enough, FRET is excited between them. The designed protein Raichu-Ras, Raichu represents a chimeric unit that binds to Ras. When Raichu-Ras was co-expressed with specific GEFs (guanine-nucleotide exchange factors) and GAPs (GTPase-activating proteins), it was clearly shown that the increase and decrease of FRET were related to the activation and inhibition of Ras mutants. They also observed Rap1 activation using the same principle.