* This product is for research use only. Not intended for use in the treatment or diagnosis of disease.
Correct subcellular protein localization is critical to the physiological environment that provides protein function. Proteins perform their biological functions in the spatiotemporal context of intact cells.
Rapid changes in the function of local proteins in cells can be achieved by specifically redirecting the localization of existing protein libraries. Eukaryotic cells have developed sophisticated targeting pathways that direct proteins to appropriate cellular locations. There are two main pathways for protein localization: nuclear and mitochondrial. The nucleus contains most of a cell's genetic material and helps to keep genetic information physically separate from other cellular functions, such as transcription and translation processes. This ensures that RNA processing (including splicing, capping, cutting/polyadenylation, and folding) will take place without intermediates being translated and interfering with the normal function of the cell.
The nuclear envelope is a double-bilayer membrane adjacent to the endoplasmic reticulum that separates the nucleus from the cytoplasm. Large polyprotein complexes called nuclear pore complexes control transport through the envelope. A nuclear pore complex composed of a class of proteins called nucleoporin can form a large macromolecular pipeline between the cytoplasm and the nucleoporin.
The transport of macromolecules into and out of the nucleus is given directionality by controlling the binding and release of cargo, which is dependent on the small GTP-binding protein/GTPase, Ran.
Nuclear genes produce most mitochondrial proteins. They will then be introduced into the mitochondria. Mitochondrial protein importation is a highly complex process due to the many functional sites in mitochondria and their bacterial sources. Protein input begins with translocation enzymes that bind mitochondrial proteins in an unfolded conformation or α-helix and move through the outer membrane complex.
The functional activities of proteins are related to their subcellular distribution and molecular complex interactions. Localization can be effectively demonstrated using fluorescence microscopy-based techniques or grading procedures. By using a recombinant reporter protein (i.e., green fluorescent protein, SNAP-tag) or a fluorescent dye, or a fluorescence-labeled molecule (protein-specific antibodies). Cells can be genetically modified to overexpress regulatory components of protein targets or non-coding sequences for purposes such as determining basic cellular processes, disease mechanisms, gene therapy, and response to therapy. Fusion protein tags can be detected via antibodies or have functional properties for localization. Bioluminescent proteins and fluorescent protein labeling systems are such genetically engineered optical imaging tools, which have higher specificity at lower concentrations than other methods. Using this method, imaging can be done in stationary or living cells, tissues, or animals. A very attractive feature of fusion protein labeling is that the labeling itself can be restricted to certain locations in the cell. This distinction is not easy to make when using bioluminescent proteins. Some fluorescent protein tagging systems cause small non-fluorescent molecules to become fluorescent when they bind to a small gene insertion peptide sequence in the target protein of interest (i.e., FlAsH). Advances in genetically engineered fluorescence systems and microscopic optical machinery have made imaging the core method for protein localization. The subcellular function of specific proteins is often analyzed through the combination of several experimental methods, such as protein purification preparation, immunoprecipitation, kinase phosphorylation, and crystal structure determination.
However, the determination of subcellular localization and function of proteins by traditional experimental methods may be a time-consuming and laborious task. Through the development of new methods in computer science and bioinformatics, coupled with an increase in data sets of proteins known to locate, computational tools can now provide rapid and accurate localization predictions for many organisms.
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