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9 주차 수업자료

9 주차 수업자료. Chapter 14. Protein Structure and Function. Secondary structure: α -helix and β -sheet. Hydrogen bond!!! Reverse turn ( β -turn): Gly, Pro. Tertiary structure. Oil drop model. Membrane protein: inverse conformation. Disulfide bond formation to maintain tertiary structure.

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9 주차 수업자료

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  1. 9주차 수업자료

  2. Chapter 14. Protein Structure and Function

  3. Secondary structure: α-helix and β-sheet Hydrogen bond!!! Reverse turn (β-turn): Gly, Pro

  4. Tertiary structure Oil drop model Membrane protein: inverse conformation

  5. Disulfide bond formation to maintain tertiary structure Disulfide bond Cysteine  Cystine

  6. Domain: independent folding region DNA

  7. Quaternary structure Hemoglobin: 4 subunits (protomers) Tetramer called apoprotein w/o cofactor Heme Self assembly (see Fig 14.15)

  8. Protein structure determination X-ray crystallography NMR spectroscopy

  9. Conjugated proteins Glycoprotein: protein + sugar group Lipoprotein: protein + lipid (e.g., proteolipid) Nucleoprotein: protein + DNA (or RNA)

  10. Function of proteins Enzymes Structural proteins Binding proteins (e.g., transporter, storage protein) Mechanical proteins (e.g., actin/myosin, chaperonin) Information processing proteins (e.g., luciferase, GFP)

  11. Characteristics of enzymes Encoded by lacZ Active site formation by protein folding

  12. Specificity of enzymes L-form only

  13. Substrate binding of enzymes • Lock and key model • Induced fit model

  14. Enzyme kinetics Activation energy required Enzymatic reaction: Vmax and Km

  15. Substrate analogs and enzyme inhibitors Substrate analog (chromogenic) Transition state analog Irreversible inhibition Competitive vs. non-competitive inhibition Reversible vs. irreversible inhibition

  16. Regulation of enzymes Negative or positive feedback Allosteric enzymes: conformational change upon binding of signal molecule

  17. Chemical modification of enzymes Phosphorylation & dephosphorylation e.g., glycogen synthesis (glycogen synthase) and breakdown (glycogen phosphorylase)

  18. DNA-binding protein (1) • Helix-turn-helix (HTH) • Helix-loop-helix (HLH) • α-helix  major groove

  19. DNA-binding protein (2) • Leucine zipper • α-helix x 2 • Hydrophobic surface (3.6 aa/turn) • Leu every 7th aa • Zinc finger (image from Wikipedia) • α-helix, β-sheet x 2 • Zn bound with 2 His and 2 Cys • Recognizes 3 bases in DNA

  20. Protein denaturation • Disruption of hydrophobic group • SDS (detergent) • Chaotropic agent • Disruption of hydrogen bond • Urea • Guanidine • Guanidinium chloride • Disruption of disulfide bond • 2-mercaptoethanol (BME)

  21. Chapter 15Proteomics: The Global Analysis of Proteins

  22. Gel electrophoresis of proteins • SDS • PAGE

  23. Antibody • Epitope • Polyclonal antibody • Monoclonal antibody • Primary antibody • Secondary antibody

  24. Western blotting

  25. Mass spectrometry • MALDI-TOF (how to: matrix-assisted laser desorption-ionization; detection time-of-flight) • ESI-MS/MS (how to: electrospray ionization; MS/MS: tandem mass spectroscopy)

  26. Protein-tagging system • His tag: His x 6  bind to Nickel ions • FLAG tag: 8 aa  anti-FLAG antibody • Avidin (streptoavidin)  bind to biotin

  27. Full-length protein fusion tag • GST (glutathione-S-transferase): bind to G-agarose • MBP (maltose-binding protein): bind to maltose/amylose

  28. Protein interactions: two hybrid system

  29. Protein interactions: co-immunoprecipitation

  30. Protein arrays

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