Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time lapsed, the proteins marked with red and green-fluorescent markers started moving laterally and were completely intermixed over the entire cell surface in 40 minutes. This experiment confirmed that the membrane proteins can show lateral diffusion.
Fluorescence recovery after photobleaching (FRAP) technique can be used to determine the diffusion rate of membrane proteins. In this technique, the target protein is marked by a fluorophore-labeled antibody which is specific to the protein. Alternatively, genetic engineering can also be used to produce the target protein fused with the green fluorescent protein. The laser beam is used to create the non-fluorescent bleached area. If 40% fluorescence recovery happens, it is considered that around 40% of the membrane proteins are laterally diffusible. The diffusion rate then can be calculated using the time required for the fluorescence recovery in the bleached area which in turn can be used to determine the diffusion coefficient. Depending upon the location and structures of the protein, each protein has a different diffusion coefficient.
The FRAP studies showed that not all membrane proteins are equally mobile, and the diffusion rate of membrane proteins is slower than that expected from the pure lipid bilayer. The rate of protein diffusion throughout the membrane is lower than the diffusion rate of membrane lipids. For example, to traverse the 20-micrometer length of a eukaryotic cell, membrane lipids take around 20 seconds, whereas membrane proteins can take up to 600-3600 seconds. Apart from the FRAP, other advanced techniques such as 1) single-particle tracking; 2) the use of optical tweezers to study the membrane barriers; and 3) genetically modified proteins to see the role of cytoplasmic and exoplasmic protein domains in the lateral diffusion have been used to explore various aspects of protein diffusion in the membrane.
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