Tutorial 3 - BCH210 Friday Tutorial Week Three David...

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BCH210 Friday Tutorial Week Three David Tulumello david.tulumello@utoronto.ca
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BCH210 Week 3 Protein Folding Questions Anfinsen’s Experiment Cardboard Box Model Protein Analysis Chromatography (ion exchange, gel, affinity, HPLC) Determination of Protein Sequence Mass Spectrometry Genetic Engineering Protein Expression/Purification SDS PAGE / Western Blot Mutagenesis of Subtilisin Catalytic Triad Mutagenesis of Met-222 Engineering of disulphide bonds First midterm in less than two weeks!
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Example Questions Which of the following in NOT a reason why an alpha- helix is a good membrane spanning structure. (a) The typical phi/psi angles of beta strands prevent them from using ATP as a form of energy. (b) Hydrophobic side chains point outwards, allowing them to face the lipid environment. (c) A 20 residue alpha helix has the same length as a typical membrane bilayer (30 Å). (d) Intra-molecular H- bonds “hide” the backbone polar sites (NH, C=O).
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Example Questions Which conclusion CANNOT be directly drawn from Anfinsen’s Experiment. (a) The primary sequence of a protein dictates it’s tertiary structure. (b) All enzymes require disulphide bonds in order to form their native structure. (c) All disulphide bonds must be between the native partners in order for ribonuclease A to function. (d) Denaturation of ribonuclease A by Urea and B-Me is a reversible process.
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Example Questions Which of the following statements concerning protein structure and folding is CORRECT. (a)Proteins typically form stable structures because all other conformations are much higher in energy. (b) Protein folding is best described as a process where all random conformations of the protein are sampled until the lowest energy “native state” is found. (c) Asn and Lys would most likely be found within the core of globular proteins. (d) Protein folding typically involves the formation of some regions of secondary structure that act to “nucleate” folding of the remaining structure.
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Protein Purification To study proteins need to isolates large amounts Must separate from other proteins/components in cells Often several steps are required to effectively purify a protein Example Start: Cell lysate (~10, 000 proteins) After gel filtration (~100 proteins) After ion exchange (~5 proteins) After affinity column (1 protein)
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Types of Chromatography Column Chromatography Gel Chromatography Separates based on size Ion Exchange Chromatography Separates based on charge Affinity Chromatography Separates based on specific interaction HPLC (High Performance Liquid Chromatography) Separates based on hydrophobicity
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Column Chromatography Used to separate proteins based on chemical properties Matrix Immobilized material in column Protein applied to top of column Gravity causes solution to pass through column Matrix (Resin) Mobile Phase Eluate
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Column Chromatography Elution profile
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This note was uploaded on 11/23/2010 for the course BCH BCH210 taught by Professor Deber during the Fall '08 term at University of Toronto- Toronto.

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Tutorial 3 - BCH210 Friday Tutorial Week Three David...

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