Of the 24,000 genes in the human genome, approximately 7,000 code for transmembrane proteins. These “macromolecular machines” are responsible for various tasks ranging from transducing signals across the lipid bilayer to allowing molecules to enter and exit the cell. They are vital for cell survival and, consequently, human health.
For instance, the BK channel, an ion channel that conducts potassium ions through cell membranes, has been implicated in the regulation of smooth muscles and neuron excitability. Defects in this channel have shown to correlate with high blood pressure and epilepsy.
However, despite the medical importance of transmembrane proteins, relatively little is known regarding the mechanisms of these “machines.” Due to insolubility in aqueous buffers as well as other problems, only 170 transmembrane proteins have been atomically mapped.
Fred Sigworth, Professor of Physiology and Biomedical Engineering, and Liguo Wang, Associate Research Scientist in Physiology, are looking to understand these elusive proteins in greater detail by employing and refining a powerful technique called electron cryomicroscopy (CryoEM).
In this technique, proteins are inserted into membrane vesicles and flash-frozen. The protein molecules are then imaged using an electron microscope, and thousands of pictures are integrated to render a 3-D map of the structure.
The advantages of CryoEM are multifold. First, compared to the older method of X-ray crystallography, which can take years to produce favorable results, CryoEM “potentially takes a fraction of the time and can provide more definite results,” says Sigworth.
CryoEM also allows one to see how different parts of the protein function. This knowledge is crucial when working with structures such as ion channels, for instance, as it allows researchers to visualize open, closed, and intermediate states.
Furthermore, CryoEM requires that the protein is implanted in an artificial membrane. Wang anticipates that this process, which ultimately mimics the natural environment of the cell, will “allow the researcher to truthfully see the object as the object itself rather than thinking about what will make the protein crystallize.” It is then possible to manipulate the environment around the protein, utilizing various stimuli to see how it affects the protein’s function.
Sigworth explains that the BK ion channel, for example, is expressed in the smooth muscle cells that make up the walls of arterioles throughout the body, contributing mainly to muscle relaxation. It works like a switch that can be turned on and off by various stimuli. When the “switch” is turned off, the muscle malfunctions, leading to ailments such as hypertension. Similarly, when BK channels in the brain malfunction, epilepsy can result.
Understanding how the parts of transmembrane proteins work is significant, notes Sigworth, as “you can make point mutations and test hypotheses about drug effectiveness and details of protein functioning.” Wang adds that this “helps the screening and optimization of drugs, as recently drugs have been aimed at structure.”
However, results thus far are not precise enough to use in medical research since only low-resolution images are available. Therefore, the next step in Sigworth and Wang’s research is improving the picture quality of the protein structure. The researchers hope to achieve this goal by obtaining more data and fitting them together more stringently. In theory, this would produce a higher resolution image that could reveal, in atomic detail, the various states that a BK channel undergoes.
Ultimately, Sigworth and Wang dream of the Yale biotechnology facility having a CryoEM service, where you can “deliver membrane protein and we’ll give the structure to you in a few weeks.”