bibc100 - Week 3 Things that denature polypeptide chain:...

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Week 3 - Things that denature polypeptide chain: Heat, extreme pH change, denaturants (urea, detergents) - Urea allows hydrophobic groups to be exposed - Hydrophobic collapse is a fancy way of saying all hydrophobic residues cluster as far away from water as they can rapidly, and it’s the most important physical property of polypeptide chain that allows folding. This first step is called molten globulin. It is very fast. Then slowly, protein folds and domains are formed. - At beginning of unfolded stage you have high entropy level, and difference of entropy is no more than a few H bonds. Protein starts to collapse, and entropy starts to minimize. At molten globular state, you get to low level of entropy. - 2 major forms of chaperones: Hsp 70 and DnaJ - Chaperones use ATP hydrolysis to help protein form into native configuration - GroEL/ES – quaternary structure of protein. Gro = bacterial virus growth mutant. Bacterial cells that were resistant to bacterial virus infection. Because bacteriaphage makes protein. When groEL missing, bacterial virus does not replicate well. - PDI can speed up protein-folding reaction - Disulfide linkages form in the ER, as this is the oxidizing area - Different types of studying proteins: o 1. Affinity chromatography – column has beads w/ligands attached, attractions of proteins to ligands filters them o 2. gel filtration/size exclusion – separates protein based on size or weight. Larger molecules come out first, smaller ones go inside porous beads. o 3. ion-exchange – separates proteins by charge. Beads attached to either an anion exchanger or a cation exchanger. Anion exchangers have a (+) charge, and cation exchangers have a (-) charge. Change pH to get protein you want out of exchangers. o 4. Isoelectric focusing - As you change the pH, you change the ionization state of all the aa in protein, but there is a continuum pH in which number of neg charges balance with number of pos charges. At that pt, you have isoelectric pt, no charge. Each protein migrates until it reaches the pH that matches its pI. Proteins with diff pI (isoelectric pts) will distribute differently throughout the gel. At high pH, all proteins are neg charged. At low pH, all proteins are pos charged. If I load the protein sample on the top here, everything will start migrate from neg to pos. the things not neg charge will go off in the other direction and isolate them. - SDS-page: It denatures proteins permanently. Heat up to 95C for 5min (very standard). Proteins denature by heat, coat by SDS. The dodecyl suflate part binds to the protein, coats with uniform neg charge. The larger the protein, the more SDS binds. But the charge/mass ratio of large protein and small protein is the same. That is why SDS-PAGE is based on the protein size not the amount of negative charges that coat the protein. Standard neg charge per mass ratio. Remember, you are eliminating the differences in proteins, whether pos or neg charges. They will all be uniformly neg charged. Add beta-ME (breaks disulfide linkages). So you will
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This note was uploaded on 10/17/2008 for the course BIBC 100 taught by Professor Nehring during the Spring '07 term at UCSD.

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bibc100 - Week 3 Things that denature polypeptide chain:...

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