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Protein Characterization I

This course is part of a series taught by Kevin Ahern at Oregon State University on General Biochemistry. For more information about online courses go to http://ecampus.oregonstate.edu/    • Плейлист   1. Folding of proteins is dictated by the sequence of amino acids. Folding does not occur randomly. If it did, proteins would take longer than the age of the universe to fold and instead they fold in seconds. This is known as Levinthal's paradox. 2. The tendency of each amino acid to participate in alpha helices, beta strands/sheets, and turns are known and this information can be used to predict secondary structure of a protein from the amino acid sequence. 3. Misfolding of proteins can have disastrous consequences (but not necessarily always). One disastrous consequence is that of prions, which are "infectious" proteins implicated in diseases, such as mad cow disease and Creutzfeld-Jacob disease in humans. Each of these is a brain-wasting disease the results from misfolding of a brain protein known as PrP. The misfolded protein apparently helps convert properly folded proteins to the misfolded state and cause the disease. 4. Cells use chaperonin complexes to help insure proteins fold properly. Chaperonins are induced by heat shock of cells. Protein Purification 1. Purification of proteins exploits differences in charge, size, shape, and affinity for specific compounds. Centrifugation (artificial gravity) provides a means of precipitating cellular components. The faster one spins the rotor of the centrifuge, the smaller the compound/structure one can precipitate. Some types of centrification, such as zonal centrifugation are used to simply separate molecules, not precipitate them. 2. Dialysis provides a means of separating large molecules (like proteins, DNA, etc) from small molecules (like salts, etc.) by encasing the protein/salt mixture in a membrane. The membrane has holes (pores) big enough to let out small molecules, but too small to let out the big molecules. Thus, with this technique, one can effectively remove the salt from a protein. 3. Gel filtration (gel exclusion chromatography) provides another way to separate large molecules from smaller ones. It employs beads with tunnels in them. The beads are packed in a column. The beads have buffer running through them and the openings to the tunnels are a fixed size (called an exclusion limit). The size of the opening determines the maximum size of molecule that can enter it. Molecules smaller than the exclusion limit enter the beads and travel a longer path than molecules larger than the exclusion limit. Thus, when large and small proteins are applied to a column the large proteins come through first and the small ones come last. 4. Ion exchange chromatography uses beads in a column also, but instead of tunnels, the beads are coated with a molecule having either a positive or negative charge. If a mixture of molecules with positive and negative charges is added to the column, the negative molecules will "stick" to the column when the beads are coated with positive charges. The molecules with positive charges will not stick to the positively charged beads and thus one can separate positive and negative molecules. 5. Affinity chromatography exploits the tendency of many proteins to 'bind' to molecules. For example, many proteins bind ATP. If one takes beads and coats them with ATP and then passes a protein mixture through the column, only those proteins that bind to ATP will stick to the column. The others will pass through it freely. Thus, one can separate proteins that bind a specific molecule from proteins that don't bind that molecule. 6. HPLC (High Performance Liquid Chromatography) employs densely packed columns containing material with chemical groups on them that interact with molecules as they are pumped through the column. The most popular type of HPLC is reverse phase chromatography, which uses column material that is very non-polar. Hydrophobic interactions cause the less polar molecules to be retained the longest by the column, whereas the polar molecules have no affinity for the column material and pass through first. 7. Electrophoresis uses electrical current to separate molecules using a gel. Agarose gels are used to separate DNAs and polyacrylamide gels are used to separate proteins. In agarose gels, DNA molecules are negatively charged, due to their phosphate backbones and are repelled away from the negative electrode towards the positive one. The speed with this they move through the gel is a function of their size, with the smallest ones being the most mobile.