Category Archives: Molecular Biology

Using Fluorescence Microscopy to Study Proteins

The approach of fluorescence microscopy has made it feasible to visualize proteins inside live cells. This is especially useful when studying the location of signaling pathways and binding partners.

Studying protein localization

The function of a protein can be assumed by its localization, particularly when a signaling pathway is known. The location of the protein is determined through the use of recombinant proteins fused with a reporter molecule, fluorescent dyes, or protein-specific fluorescent antibodies.

Fluorescent proteins recombined with either target protein or regulatory regions can be overexpressed inside cells to know fundamental cellular processes. An advantage of using genetic systems to express proteins is that the fluorescent proteins can be selectively activated in specific regions of tissues or cells. This makes the process of visualizing proteins easy.

Studying protein structure

The techniques earlier applied to analyze protein structure include cryo-electron microscopy, protein NMR, and X-ray crystallography. In cryo-electron microscopy, high energy electrons are focused at the sample to form a 2D projection. After capturing the 2D orientation of molecules from all angles, a 3D image is developed. However, this process cannot be performed in live samples and it requires large sample preparation.

Alternatively, nuclear magnetic resonance (NMR) captures the magnetic and chemical properties of the atoms but requires a pure solution of the sample which is again not optimal for studying cells. X-ray crystallography needs the sample to be crystallized before it can be imaged. In this case, the process of crystallization can modify the native conformation of proteins.

Contradictory, super-resolution methods, such as photoactivated localization microscopy (PALM) or stimulated emission depletion microscopy (STED) have a high resolution of 20-30 nanometers. This is below the diffraction limit of conventional microscopes which is 250 nanometers. Using these techniques, protein and membrane structure can be visualized to a great detail in their in vivo surroundings.

Studying protein dynamics

Using fluorescent recovery after photobleaching or FRAP, it can be measured how a protein moves or diffuses inside a cell. The diffusion of a protein is associated with its folded or misfolded status, its stability, and binding to other molecules. Thus, understanding its movement or diffusion can tell us its structure and function inside cells.

In this process, the fluorescent molecules in a specific region of a cell are completely bleached by shining an intense and constant beam of light. It results in the recovery of the fluorescence in the bleached region is measured as a function of time to map its diffusion parameters.

Studying protein signaling and interaction

This is defined using a fluorescence method called Forster resonance energy transfer or FRET. This technique is based on the transfer of energy between two closely placed molecules conveniently called donor and acceptor molecules.

The donor chromophore is originally in an excited state and as it is close in proximity to another molecule, it transfers its energy to a donor chromophore. This transfer is proportional to the sixth power of the distance between donor and acceptor.

Hence, based on the efficiency of the transfer of energy, the distance between two molecules or protein domains can be concluded. Thus, allowing scientists to determine which protein domains are interacting during a signaling event.

Studying protein levels

Apart from signaling, structure, and function, the fluorescence of a protein can also be utilized to determine the levels of a protein expressed inside a cell or a tissue. It is mostly used in cases where the fluorescent probe is recombined with the gene or reporter of a gene. The expression of the reporter reflects the levels of protein it is recombined with.

Scientists Reveal Structure of Amino Acid Transporter Involved in Cancer

A team of scientists have used cryo-electron microscopy to elucidate the structure of the protein, which may generate leads for drug (12)

The human glutamine transporter ASCT2 is increased in several forms of cancer. It is the docking platform for a wide range of pathogenic retroviruses. The ASCT2 protein imports the amino acid glutamine in the human cells and maintains the amino acid balance in many tissues. The amount of ASCT2 is increased in several types of cancer usually because of an increased demand for glutamine. Moreover, several types of retrovirus infect human cells by first docking on this protein.


ASCT2 belongs to a larger family of similar transporters. To understand the working of this family of amino acid transporters and to help in designing the drugs that block glutamine transport by ASCT2 or its role as a viral docking station the scientists have resolved the 3D structure of the protein. They resorted to the technique of single particle cryo-electron microscopy, as they did not succeed in growing crystals from the protein, which are required for X-ray diffraction studies. The human gene for ASCT2 was expressed in yeast cells and the human protein was purified for (11)

The structure was determined at a resolution of 3.85 Å revealing the striking new insights. It was a challenging target for scientist as it is rather small for cryo-EM. But it also has a good symmetric trimeric structure, which helps.


The cryo-EM images reveal a familiar type of ‘lift-structure’, where a part of the protein travels up and down through the cell membrane. The substrate enters the lift in the upper position and then moves down to release the substrate inside the cell. The structure of ASCT2 depicts the lift in the lower position. It had been thought that the substrate enters and leaves the lift through different openings, but the results suggest it might well use the same opening.

This information could help design molecules that stop glutamine transport by ASCT2. Some tests in mice with small molecules that block transport have been published. Blocking glutamine transport would be a way to kill cancer cells. This new structure allows for a more rational design of transport inhibitors.


Another observation was the surprise for the scientists as they observed the spikes that protrude on the outside of each of the three monomers. These are the places where retroviruses dock. This proved to be consistent with mutagenic studies performed by others. By knowing the shape of the spikes could help design molecules which block the viruses from docking. The protein structure was resolved in some time duration, which is remarkably fast for cryo-EM.

Future studies will be done to capture ASCT2 in different configurations, like inside a lipid bilayer rather than the detergent micelles used in the present study and with the lift in different positions. Thus, it was concluded that studying different states will help them understand how this protein functions.