BIOL 302 1st Edition Lecture 7 Protein Misfolding and Sequence DeterminationOutline of Last Lecture I. Chemical bonds II. Protein Structures III. Protein Folding, Unfolding and MIs-folding Outline of Current Lecture I. Protein Misfolding II. Protein Sequence DeterminationCurrent LectureIII. Pathogenic ProteinsA. Protein misfolding can cause disease and certain misfolded proteins, called prions, can cause diseases that are infectiousB. The existence of prions showed that heritable biological information can be enciphered in a molecule other than nucleic acidIV. Prion Diseases (Proteinaceous infectious only protein (PrP))A. Creutzfeldt-Jakob disease shows spongiform (vacuolar) degeneration, the most characteristic neurohistological feature; in cerebral cortexB. Forms amyloid fibers in brain scrapie, mad cow diseaseV. What prevents unfolded proteins from forming pathogenic aggregates? Why are they toxic?These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.A. http://ed.ted.com/lessons/the-coming-neurological-epidemic-gregory-petskoVI. iClicker question: Which of the following is NOT true about the Unfolded protein response? A. The unfolded protein response is turned on by protein misfolding.B. The size of the endoplasmatic reticulum is decreased to reduce protein synthesis.C. Chaperones will be produced in larger quantities to aid protein folding.D. Ire1 acts as receptor to transmit signals that regulate the responseThe size of the ER is increased to increase the capacity of the cell to process secretory proteins and to aid proper protein folding.Protein Purification and CharacterizationVII. Purification is the first step in understanding protein functionA. The goal of purification is to provide an aqueous sample containing only one type of molecule – the protein of interest.B. Techniques of separation make use of proteins size, shape, charge, and when possible, specific binding “tags”C. The approach is to always minimize the number of purification steps and maximize the product yield in the most cost effective wayD. Techniques used at the early stages show high capacity, speed and low cost, but only modestselectivity. E. At late stages, high resolution is the goal; therefore higher expense and low capacity can be accepted because sample volumes are usually smallerVIII. AssayA. RAPID QUANTITATIVE SPECIFIC SENSITIVEB. Used to monitor protein purificationC. Critical to any efficient purification is a specific and sensitive AssayD. Specific means to identify protein of interest within a complex mixtureE. Allows you to track your protein and analyze purification efficiencyF. In the reaction of lactate with lactate dehydrogenase to produce pyruvate NADH and H, the enzyme has a different absorbance than the substrate. The increase in absorbance of NADH at 340 nm is used to follow the formation of pyruvateIX. Assessing purification with SDS-PAGEA. SDS Gel electrophoresis1. Make gel, separate proteins according to size, stain gel, see protein bands with different sizes, with each purification step we eliminate more and more bands to get target proteins of size we predicted to have2. Proteins can be separated by electrophoresis and stained with Coomassie blue3. Electrophoretic mobility of a particular protein is a function of the ratio of its charge to its frictional coefficient (shape)4. Not super specific because we can have multiple proteins of the same size…which is whywe need to continue with PAGEB. SDS-PAGE1. Polyacrylamide gel electrophoresis (PAGE)2. Acrylamide mixed with bisacrylamide forms a cross-linked polymer network when the polymerized3. SDS at a given solution concentration binds to many proteins at a constant weight-weight ratio. 4. This means there is a defined number of bound SDS molecules per amino acid residue.5. Therefore, the charge contributed by SDS will greatly outweigh the intrinsic charge of the protein and the charge contributed by SDS is proportional to protein molecular weight6. The wells at the top are filled with the samples and it migrates down towards the positively charged bottom7. Migration is proportional to the log of the molecular weight of the protein8. SDS-PAGE separates proteins by sizea. When we denature, shape will change, no bonds, no native charge, tiny contribution of other factors=mainly separated by size9. Native PAGE (non-denaturating conditions, without SDS or reductant) separates proteinsby: a. Chargeb. Sizec. Shaped. Oligomeric stateX. Proteins must be released from the cell to be purifiedA. Mechanical and chemical means are used to disrupt cells containing our protein of interestB. HomogenizationC. Blender, French Press, SonicatorXI. Proteins can be fractionated by centrifugationA. Following lysis, the homogenate can be fractionated by centrifugationB. As the homogenate is spun, dense heavy material sediments at the tube bottom, forming a “pellet”XII. Salting out – (NH4)2 SO4A. Most proteins are less soluble at high salt concentrationB. Ammonium sulfate is the most common salt used:1. Because it is unusually soluble in cold buffers 2. Because it yields a precipitated protein slurry that is usually very stable, so the purification can be stopped for a few hours, or even daysC. Salt concentrations of 1-2 M are not unusualD. Low salt – (0.1 to 0.5 M) increasing the ionic strength shields unfavorable electrostatic interactions, thereby increasing protein solubility E. High salt –At elevated concentrations of salt (>1 M), much of the water that would solvate the protein is taken up in the hydration shells of the ions. Without that water shell, the protein precipitates out of solutionXIII. DialysisA. Removing salt from a solution of protein is done by dialysisB. Take protein with salt, put into dialysis membrane with holes to allow the exchange of waterC. Ammonium sulfate>protein is at same concentration, water goes in to dilute, ions go into buffer outside of the dialysis tubing, same concentration of salt in membrane and buffer because of Le Chatleier's PrincipleD. IClicker Question: If you dialyze a 1 ml sample of protein in 1.0 M (NH4)2SO4 against 1 L of water, what is the final concentration of (NH4)2SO4 in your protein sample?1. 0.01 mM2. 0.1 mM3. 1 mM4. 10 mMXIV. Ion-exchange chromatographyA. Cation and anion exchange chromatography exploits variations in charge density