Category Archives: Structural 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.

Tiny straws improve molecule delivery

Researchers have found what they believe may be a better way to deliver molecules directly into human cells they require to manipulate. “Nanostraws” are small glass-like protrusions that poke equally little holes in cell walls before releasing their cargo that is completed more safely and with efficiency than existing strategies.

They say breakthrough might speed up medical and biological research and in the future will improve gene therapy for cancer and diseases of the eyes and immune systems. Scientist expertise with nano materials was the key to the new approach,that improves on the method referred to as electroporation, whereby an electrical current is employed to form holes in cell walls.

Existing electroporation strategies will be imprecise, usually killing several of the cells researchers try to work with. Nanostraws are more economical as a result of their long, narrow profile helps concentrate electrical currents into a really tiny space.

They’re additionally a far better possibility than approaches based mostly around using viruses or alternative chemicals to carry molecules across cell walls. The researchers initially tested their technique on animal cells sitting atop a bed of nanostraws. when they turned on the electrical current, the nanostraws opened little, regularly sized pores within the cell membrane: enough to permit molecules in, however not enough to try to to serious injury. the current drew molecules straight into the cell, further increasing the speed and precision of the method.

The question at that time was whether or not the technique would be as effective on the kinds of human cells clinicians would wish to manipulate to treat diseases. They successfully delivered molecules into 3 human cell types as well as mouse brain cells, all of that had proven difficult to work with in the past. The technique was fast and killed fewer than 100% of cells, an enormous improvement on normal electroporation, they say.

The next step is to test it with human immune cells,that are among the hardest to figure with.

Taking a closer look

Cryo-electron microscopy,an emerging technology that can show the structure of molecules down to the atomic level with more clarity than ever before. Years ago, we were able to see only the general areas where the proteins were as blobs on a screen but now it is possible to see the atoms within those blobs. This could help researchers design new drugs that better target diseases or create new compounds that speed up or slow down chemical reactions.

Electron microscopes use a beam of electrons instead of rays of light to create an image. Because electrons have a much shorter wavelength than visible light, such microscopes can provide better resolution of molecules at the atomic level.

What we are looking at is a projection of how the electrons interact with the sample. Since everything is made of electrons, they repel each other. Thus, what is generating is a sort of two-dimensional projected image of your sample.

Cryo-EM is a modification of the standard electron microscope that involves freezing a sample in solution so rapidly that the surrounding water forms a thin, glass-like layer instead of crystallizing. Scientists can then take multiple images of that frozen sample and piece them together to create a remarkably clear 3-D image.

Seeing the shape and arrangement of the molecule could help scientists better understand how those molecules work. Some proteins operate very much like tiny machines, using a chemical process to create mechanical energy. They have very mechanical properties, so when we see the whole thing, then we immediately start to see what the pieces are and how they move. With cryo-EM scientists can take various images of these proteins and put them together like a stop-action movie to see how those mechanical properties work.

As the technology has improved it’s opened up new areas of research. Pharmaceutical companies, for example, are using cryo-EM to map out the sites where drugs can bind to the target cells, so they can better identify which drugs might affect those cells and learn what part of the protein they interact with.