Fluorescent proteins: why molecules glow

Operating principle

Every resident of the metropolis knows perfectly well how to use contactless bank cards or travel cards and similar passes to school or work. It couldn’t be simpler: bring your card to the turnstile, the green light comes on - go through.

The principle of operation of such a device is clear even to a schoolchild: there is a transmitter in the “head” of the turnstile, and a small receiving device in the card. If this device is brought to a certain distance from the turnstile transmitter (in the case of the subway, this is a few centimeters), it receives the radiation from the transmitter. An electric current arises in their interaction circuit, and the simple electronics of both devices begin to do their job - checking the month and date, the number of trips. The same thing happens with a bank card brought to the reader, only the data array increases slightly. That is, a ticket or card receives energy from the reading device, and then emits it, and the energy of the ticket or card, according to the law of conservation of energy, will always be less than that which they received from the reading device, because part of it went to processing information and at the same time heated its environment (literally). It is clear that this interaction occurs only if the transmitter and receiver are at a certain, fairly close distance.

But we should be concerned about another question: how small can these two devices be made? Today the answer is: the size of a molecule or even smaller, but in this case the transmitter and reader molecules must be fluorophores: one must be able to emit light (donor), and the second must be able to absorb it (acceptor). Moreover, such energy transfer is possible not for any, but for strictly defined pairs of donors and acceptors (you won’t get into the subway using your work pass).

It is clear that in the case of very small transmitters, the interaction between them occurs at distances equal to the intermolecular interaction - several tens of angstroms.

Interestingly, fluorophore molecules do not interact with each other in exactly the same way as other molecules. By emitting energy, they slightly heat up all the solvent molecules (the same happens in the case of a travel ticket or card), but when they meet their fluorophore-acceptor, the donor transfers energy to it without radiation, however, the acceptor goes into an excited state and now capable of emitting light itself. In addition, in both the contactless operation of the card and the energy transfer between fluorophores, the distance at which the interaction occurs is significantly less than the wavelength of the radiation that the transmitter emits (it does not matter whether it is a turnstile or a donor molecule). The main requirement for the “donor ⎼ acceptor” pair is spectral overlap, that is, the donor emits light only at those wavelengths that the acceptor can absorb, but energy transfer occurs without photon emission. This phenomenon is called FRET (Fluorescens Resonans Energy Transfer - resonant fluorescence energy transfer) (Fig. 1).

Fig. 1 Physical nature of the fluorophore

Resonant fluorescence energy transfer

This phenomenon has features that have made it an indispensable research tool in molecular biology and biophysics. When the donor and acceptor approach each other, energy transfer occurs inevitably; this fact makes it possible to use energy transfer as a sensor for the approach of two molecules in space. To do this, it is enough to place fluorescent labels on two molecules.

This versatility allows us to use this method to study the interaction of any molecules (DNA, proteins, peptide regulators, growth factors, hormones) and, for example, cell membranes or intracellular structures, since by selecting fluorophores and modifying molecules with them, we turn on the alarm. The appearance of energy transfer, which can be observed as a fluorescent signal in a microscope, will be tantamount to its activation. Moreover, it is not at all necessary to attach a chromophore to proteins; many of them already have fluorescent properties; many cells themselves synthesize fluorescent proteins, so their damage during research inside a living organism can be minimized or eliminated altogether.

A little history
The history of the discovery of fluorescent proteins is interesting. Back in 1955, the discoverer of green fluorescent protein, Osamo Shimamura, as a student at Nagoya University, began studying bioluminescence and tried to understand why decaying mollusks of the genus Cypridina glow in the dark. At that time, Osamo was not even a graduate student yet, but his success impressed the professors so much that he received a PhD (in our classification of academic degrees - candidate of sciences) in absentia, and after the publication of the results he was invited to work in the USA. There, Shimamura continued to study luminous organisms, collecting and processing up to three thousand jellyfish on the seashore. The first fluorescent protein isolated from jellyfish was aequorin. This is a protein that did not glow green, like the jellyfish themselves, but with blue light, which is due to the oxidative addition of the coelenterazine molecule and the presence of calcium ions in sea water. This aequorin-coelenterazine system is still used as the main biochemical sensors for the presence of Ca 2+ molecules in solution. Do you get the idea? In order to find a molecule in a solution, it is enough to color the solution, and the number of molecules can be counted by the intensity of the glow.

In 1962, Shimamura realized that the jellyfish contains another fluorescent protein and for its glow only an exciting stream of light is needed, oxygen in normal concentrations and, unlike its “firstborn” aequorin, it does not require any additional cofactors. This green fluorescent, called GFP, was awarded the Nobel Prize 50 years later. In those distant times, this phenomenon was difficult to use; these were only the first steps in biotechnology.

The revolution began when Shimamura, after meeting at Columbia University in New York with Martin Shelfi, who was studying the famous worm Caenorhabditis elegans, a favorite of geneticists, proposed using GFP to map the cells of the developing embryos of this worm, as well as to study some intracellular proteins. This became possible thanks to the rapid growth of technology and technical capabilities in the late 80s of the last century. The results of their research amazed scientists all over the world: inside the cell it was possible to monitor the movement of individual proteins in real time, the body practically did not suffer from this. And most importantly, this required an ordinary microscope with an ultraviolet lamp and a light filter that would transmit the required wavelength. In Figure 2, we see that activation of cells by TNF-a causes a circular movement (cytoplasm ⎼ nucleus ⎼ cytoplasm) of transcription factors (NFkB, green) and their inhibitors (IkB, red). The numbers in the corner indicate the time in minutes after stimulation, and the arrows indicate the same cell.

Rice. 2. Activation of cells by TNF-a causes a circular movement “cytoplasm - nucleus - cytoplasm” of transcription factors (NFkB, green) and their inhibitors (IkB, red)

It is worth paying attention to the red color in this picture. It turned out that using targeted mutagenesis it is possible not only to enhance the luminescence of a protein, but also to change its color. This is how yellow, red, and blue variants of GFP turned out. It is also important that in order for proteins to have fluorescence, it is not necessary to chemically attach a fluorophore to them; you can simply use proteins that already have this property and glow, like jellyfish and other marine life. That is, the luminous protein can be synthesized by the cell itself, which means that the impact on it from the outside and damage is minimal.

Why do we need all this?

What do we want to install such a unique color fluorescent alarm on? The answer will depend on each unique scientific problem: for example, by tagging an enzyme and the substrate it acts on with fluorophores, we can monitor when and under what conditions (time, pH, temperature, concentration) they interact, sometimes directly in a living organism and in real time. Isn't this one of the wonders of modern science?

Using fluorescent mitochondria as an example, we will be able to examine and analyze their work, and in the future see the delivery of genetic constructs - parts of DNA into the cell ⎼ and visualize this miracle of genetic engineering (Fig. 3).

Rice. 3. Delivery of genetic constructs into human cells

Another important aspect of using the described phenomenon is that the efficiency of energy transfer depends on the distance between the fluorophores (remember the card and the turnstile). To be more precise, it is inversely proportional to the distance to the sixth power (r-6), and if this is so, then energy transfer is very convenient to use for measuring the distance between molecules, organelles, enzymes and their substrates, antigen proteins and antibodies and other microscopic objects . This spectroscopic tape measure with a measurement range of several angstroms will allow one to determine, for example, how close two proteins on the surface of a cell membrane are to each other, as well as any value derived from it - the adhesion force of the cell to the surface on which it lies. Can you imagine what this means for a child born with Lyell's syndrome? The degree of adhesion of keratinocytes to the basement membrane can be very accurately determined, and this will clearly allow one to assess the severity of the disease and, accordingly, the prognosis.

Once again, I would like to draw attention to the fact that optical microscopy with its resolution has previously made it possible to study the morphology of a cell, but the limit of this resolution did not allow us to look deeper and examine organelles and individual molecules. For this, it was possible to use harder ⎼ for example, short-wave radiation during electron microscopy, but it did not allow working with living objects, but only with fixed, dead cells. And only now can we look deep into ourselves without harming our health. In addition, by coloring two proteins in different colors, it is possible to trace all the nuances of their interaction and their distribution in the cell or see how predator vibrios kill prey vibrios (Fig. 4), assessing the effectiveness of autophagy as a not entirely new method, but one that has become relevant modern therapy of infectious and viral diseases due to the fact that we are entering a dangerous era of ineffective antibiotics.

Rice. 4. Predator vibrios kill prey vibrios

Rice. 5. Chimeric mice expressing RFP (red fluorescent protein) are used to study patterns of individual development

In addition, some tumors themselves can synthesize fluorescent proteins (Fig. 5), and those that cannot, we can completely safely stain them, and also add fireflies that signal diseases. Soon a gadget will probably be created that will be located somewhere in our body, and the remote control for it will be in the hands of our doctor. And even on the other side of the continent, by changing the color signal from our body, he will be able to call us for examination and treatment.

First published: Les Nouvelles Esthétiques Ukraine" No. 1 (101)/2017