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.