Protein Constructor
Biologists have learned how to quickly reassemble protein biosensors for the desired pathogen
Biologists have created a class of protein biosensors that glow in the presence of a given pathogen or disease marker. According to the researchers, the modularity of the system makes it possible to quickly create biosensors for new target molecules: it is enough to conduct a computer analysis and replace one of the protein domains. The authors of the work published in Nature (Quijano-Rubio et al., De novo design of modular and tunable protein biosensors) tested the sensor they described on botulinum toxin, coronavirus, a marker of myocardial infarction and other clinically significant targets.
Protein-based biosensors play an important role in synthetic biology. In basic research, protein sensors are used to study the processes taking place in cells, and in medicine, for example, they hope to predict the effectiveness of therapy for patients with cancer with their help. Such sensors are created on the basis of proteins existing in nature. To do this, a protein domain is selected that can bind to the target molecule, and connects to the reporter domain, which produces a readable signal. The main disadvantage of this approach is labor–intensive, because it takes a lot of bioengineering transformations to get an effective biosensor from these two domains. Therefore, researchers are looking for a universal platform on the basis of which it will be possible to easily create biosensors for protein molecules of various pathogens or disease markers, simply replacing the necessary "details" in it. Rational computer protein design, which has been actively developing in recent years, makes it possible to create proteins with the necessary de novo characteristics.
A protein system with two almost equal energy states can serve as a biosensor. And the presence of the target molecule should regulate whether the reaction will proceed in the direction of a visible response, for example, glow or color change.
Scientists distinguish three requirements for a potential sensor system: a change in the system should lead to a visible result; a change in the system that leads to a visible result should not depend on the target molecule so that the system as a whole can be used to detect different substances; and, finally, the system should be easily configurable so that it can It was necessary to detect substances with different energy characteristics of binding to the sensor, and so that the minimum detectable concentration could be adjusted.
Researchers from The University of Washington, led by David Baker, has created a protein system that meets these requirements. Biologists called the components of the system quite simply: the cell and the key. Both parts contain a piece of luciferase, and only when the key is attached to the cell, the luciferase becomes whole and triggers a glow reaction. There are two domains in the cell: the cell itself and the latch connected to it. The latch covers the place where the key should join. In the presence of the target molecule, the latch binds to it, freeing up space for the key, and the emitted glow indicates that the reaction was successful.
The system is designed in such a way that the energy of binding the key to the cell is not enough to exceed the energy costs of opening the cell in the absence of a target. But in the presence of a target, the additional binding energy of the target to the latch causes the latch to open and leads to a glow. Since the key and the cage are always the same, the system can be rebuilt for different targets by simply changing its binding domain in the latch. The functioning of the system depends on thermodynamic equilibrium, therefore, by setting the necessary binding energies in the system, it is possible to regulate the minimally detectable concentrations of substances.
The system was tested for botulinum toxin, a toxic substance, a neurotoxin that is produced by anaerobic bacteria and often causes serious poisoning with canned food. The ingestion of botulinum toxin in the body with food causes severe disorders in the nervous system and paralysis of skeletal muscles. An instant determination of the cause of poisoning would help to provide the necessary assistance faster, and preventive food testing would prevent such cases. Researchers have shown that the response to the presence of botulinum toxin is visible after a few minutes. In a similar way, the authors determined the presence of the HER2 protein in the solution, the increased expression of which in humans indicates the development of certain types of breast cancer, and a marker of myocardial infarction.
Scientists have not passed by the coronavirus pandemic: they showed the possibility of detecting antibodies to covid and the spike protein of the virus, which it uses to enter the cell.
It was also possible to detect the presence of hepatitis B virus and antibodies to it.
The authors of the work note that their development should be convenient for clinical use: the entire reaction takes place in one test tube, the result is visible almost instantly, and it can be accurately assessed using a simple mobile phone camera. De novo created proteins are well developed by cells, so there should be no problems with mass production of biosensors.
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