Naked proteins
Proteins are essential for life, since they participate in most of the functions performed by the cells of our body. Moreover, diseases are directly related to proteins, as they are caused by proteins that function in a way or time that they do not need.
At the CICbioGune research centre, proteins are observed to determine their structure. With the knowledge of the form, it may be possible to develop drugs that can interfere with the functioning of proteins.
Proteins are involved in most of the cell functions of the human body, so they are vital to life.
The main task of the researchers of the Department of Structural Biology of CIC bioGUNE is precisely to study proteins, to find their structure.
But for what?
Adriana Rojas, CIC bioGUNE
“If you have a disease, it has something to do with a protein that doesn’t work well or works when you don’t need it. The function of the protein is related to the three-dimensional shape of the protein. If we understand the shape of proteins, we can design compounds that will stick to the protein so it works when it needs to work or stay when it needs to work.”
Knowledge of the structure of proteins can thus become a tool for the development of new healing pathways.
Of course, in order to study proteins, they must first be purchased.
At CIC bioGUNE, genetically modified bacteria are used to acquire the protein to be investigated. First, the culture liquid in which the bacteria are to grow is prepared, then the appropriate conditions for proliferation are provided: food, required temperature, and oxygen.
Various techniques may be used to extract proteins from bacteria. This device, for example, breaks the membrane of bacterial cells using ultrasound. In this way, proteins are released.
There are a number of techniques available for the analysis of proteins using a combination of several techniques. One of them is crystallography: X-ray diffraction that provides high-definition information about the structure of proteins.
In order to use this technique, it is necessary to crystallize the proteins.
Adriana Rojas, CIC bioGUNE
“I put the solution underneath and the protein and another solution in the drop. I close the system and wait for the sample to concentrate over time and form the crystal, as happens when the water freezes. By changing a variable, the temperature, the water turns into ice. Here, I move other variables so that the protein is organized into crystals. I change the pH, the temperature, the salt concentration... and in order to do all this, I have to do a lot of tests.”
Crystallization robots are used for this purpose. The robots use nanoliter samples and make thousands of combinations automatically, until in a few of them the proteins become crystals.
Adriana Rojas, CIC bioGUNE
“Through the robot, I take samples from this main assembly. The robot comes, absorbs the solution—and each solution is different—passes it to this set, where I already have 96 tests. When it leaves this on the plate, the robot leaves the solution underneath. I already have half the test, and now I have to pass the protein. To do this, I use this other robot that distributes protein. This robot can deliver up to 50 nanoliters of protein, which is very little. So I can do 100 tests with 15 microliters of protein, whereas I could have done 15 before.”
This is a crystal culture, where crystals can grow at a temperature of 21 degrees Celsius. In order to be able to keep track of the proteins, each plate has a code and a photograph of each drop is taken periodically.
When the crystals are large, they can be taken directly and exposed to X-rays, but when they are small, it is necessary to repeat the test, but with a larger amount.
Adriana Rojas, CIC bioGUNE
“I can scale the test to larger drops. For what purpose? To manipulate the crystals more easily, because I have to pull the crystals out of the drop, I have to fish them and freeze them in nitrogen to take them to the X-rays.”
The crystals are inserted into the x-ray machine and subjected to an x-ray. Because they are symmetrically arranged, the proteins create molecular planes that work as mirrors, thereby changing the direction of the X-ray beam as it hits the crystal. With computerized mathematical calculations, the structure of the protein is determined.
The Magnetic Resonance Room is one of the most picturesque facilities at CIC bioGUNE. The building was built and then the installation was built.
Oscar Millet, CIC bioGUNE
“We are now underground because these devices are on the ground. Each of them has a concrete column, completely separated from the foundation of the building so that vibrations can be avoided.”
The temperature is also controlled, and the fluctuations cannot exceed half a degree; humidity, carbon dioxide, etc. are also controlled.
These resonance devices basically use the same technique as clinical diagnostic devices, but applied to very small things.
Tammo Diercks, CIC bioGUNE
“This is the only technology that allows us to study proteins or molecules and their structures with resolution at the atomic level and in solution, in the liquid state. This is a large container filled with cryogenic material: it is liquid nitrogen from the outside at a temperature of -198 degrees centigrade. This is insufficient to achieve the superconductivity of the coil, the core of the magnet. Then, inside the liquid nitrogen-containing container, there is another liquid helium-containing container. This cools down to -270 degrees, allowing the coil contained therein to be superconducting. In this way, we can obtain a magnetic field, very stable, strong and homogeneous.”
Oscar Millet, CIC bioGUNE
“Each of the atoms in the spectrum is located according to what surrounds it; that is, it tells us who it is and also where it is located. For example, here we have an image of a protein and several signals. Each of these signals corresponds to an atom of one of the amino acids. Proteins are made up of amino acids and amino acids are made up of atoms. Depending on where they are located, we know which amino acid it is, which amino acid is next to it, and which amino acid may be near it. So it gives us information about the structure. When we have the structure model, it’s easy for us to know, for example, how our protein interacts with a drug.”
Electron microscopy completes the trio of protein observation techniques at CIC bioGUNE.
Mikel Valle, CIC bioGUNE
“In our group, we used the microscope to calculate the three-dimensional structure of proteins and other biological molecules. At the top of the microscope we have a cannon that emits electrons; the electrons pass through the entire column of the microscope, including our molecules at this height, and we acquire two-dimensional images. From these two-dimensional images, using computation and image processing, we can obtain three-dimensional models like this. In this case, it is a three-dimensional model of a ribosome. Ribosomes are plants that synthesize proteins inside the cell, and we want to know how ribosomes, placed under different conditions, work to know how they work.”
Combinations of structures are made in electron microscopy and observations can be made about the dynamic appearance of proteins. They are very close to what is happening in reality.
With knowledge of the structure, it is possible to know how the protein works and design drugs to activate or inhibit it. It can be an effective strategy to develop new healing pathways.
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