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SURVEY OF BIOCHEMISTRY Proteins and Biomolecular Stability

SURVEY OF BIOCHEMISTRY Proteins and Biomolecular Stability. Protein Structure. Primary (1°): amino acid sequence Secondary (2°) Alpha Helix Beta Sheet Tertiary (3°) Quaternary (4°). Primary and Secondary Structure. Superoxide Dismutase - 1XSO. Myoglobin - 2V1K. Recap.

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SURVEY OF BIOCHEMISTRY Proteins and Biomolecular Stability

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  1. SURVEY OF BIOCHEMISTRYProteins and Biomolecular Stability

  2. Protein Structure • Primary (1°): amino acid sequence • Secondary (2°) • Alpha Helix • Beta Sheet • Tertiary (3°) • Quaternary (4°)

  3. Primary and Secondary Structure Superoxide Dismutase - 1XSO Myoglobin - 2V1K

  4. Recap • Structures of 20 amino acids • pKa and pI • 1°: Polypeptide Sequence • 2°: Secondary Structures • Alpha Helices • Beta Sheets

  5. Recap continued • Protein Purification Methods • Gel Filtration • Ion Exchange • Affinity • How to assess purification? • Purity • Yield

  6. SDS-PAGE • Electrophoresis: a method for separating molecules based on size and charge when exposed to an electric field. Name “SDS-PAGE”: SDS = sodium dodecyl sulfate PAGE = polyacrylamide gel electrophoresis

  7. Sodium Dodecyl Sulfate SDS confers negative charge on proteins and denatures proteins Amphiphilic Anionic Detergent in a wide variety of products Hydrophilic Hydrophobic Sodium Dodecyl Sulfate(Lauryl Sulfate) Proteins are primarily denatured by boiling them prior to electrophoresis! CH3(CH2)11OSO3

  8. SDS-PAGE Buffers Buffers maintain pH control Allow gel to fully polymerize Stacking Gel 0.5M Tris-HClpH 6.8 Resolving Gel 1.5M Tris-HClpH 8.8

  9. Sample Preparation Ensure that sample has fully dentured! In the Gel In the Sample Stacking Gel 0.5M Tris-HClpH 6.8 Laemmli Sample Buffer 0.5M Tris-HCl,pH 6.8 SDS Glycerol Bromophenol Blue Resolving Gel 1.5M Tris-HClpH 8.8 Boil Sample for 1-5 min

  10. + + H3N-CH2-COOH H3N-CH2-COO - Electrophoresis Buffer Electrophoresis Buffer Tris Base, Glycine, SDS FullyProtonated Loss of 1Proton Loss of 2Protons - H2N-CH2-COO AcidicForm ZwitterionicForm BasicForm

  11. SDS-PAGE In the Gel Gly lags Stacking Gel 0.5M Tris-HClpH 6.8 - + H3N-CH2-COO Zwitterion Form Gly leads - Resolving Gel 1.5M Tris-HClpH 8.8 H2N-CH2-COO Basic Form Note: Discontinuous SDS-PAGE is depicted here!

  12. Migration in an SDS-PAGE Gel - +

  13. Migration in an SDS-PAGE Gel Stop electrophoresis when dye front reaches bottom of gel Stain with Coomassie

  14. Purity • Purity is a measure of how undefiled a protein sample is. Pure protein Lots of impurities

  15. Amount of protein recovered % Yield = Amount of protein initially Yield Example: 1 2 3 After 2 steps of purification % Yield = (208 / 358.2) x 100 = 58.1%

  16. Protein Sequencing Study how each works! • Separate subunits • Dansyl Chloride Reaction • Proteolytic Digestion • Cyanogen Bromide Cleavage • Edman Degradation Read on yourown!

  17. How to separate subunits? Dithiothreitol, DTT 2-Mercaptoethanol

  18. Proteolytic Digestion Know this!

  19. Proteolytic Digestion How many fragments would result from digestion with trypsin?

  20. Proteolytic Digestion Trypsin cleaves after Lys (K) and Arg (R): 16 fragments!

  21. Protein Structure Classifications • 1°: amino acid sequence • 2°: local spatial arrangement of a polypeptide backbone without regard for side chains • 3°: 3D structure of a protein including its side chains • 4°: spatial arrangements of subunits

  22. Beta Tertiary Folds Some proteins only have alpha helices (plus turns and random coils). Others only have beta sheets (plus turns and random coils). Alpha

  23. Alpha/Beta Tertiary Folds Some proteins have a combination of alpha helices and beta sheets(plus turns and random coils).

  24. Biomolecular Stability Nucleic acids and proteins are stabilized by the same types of intermolecular forces. Hydrophobic Effect: the tendency of water to minimize its contact with hydrophobic groups in molecules. How does the hydrophobic effect impact proteins and nucleic acids?

  25. Entropy Entropy measures the spontaneous dispersal of energy: how much energy is spread out in a process -or- how widely spread out it becomes — at a specific temperature http://www.entropysite.com

  26. Entropy & the Hydrophobic Effect How is entropy increased by the hydrophobic effect? http://www.cryst.bbk.ac.uk/PPS2/projects/day/TDayDiss/Major.html

  27. How do bond energies compare? Type of Bond Bond Strength (kJ/mol) Covalent 348 - 460 Ionic Interaction 86 Hydrogen Bond 20 Dipole-dipole 9.3 London Dispersion 0.3 Table 2-1 Relatively speaking, H-bonds are weak,but they are not nearly as weak as one might expect!

  28. Why Do Base Pairs Stack? ENTROPY Hydrophobic effect induces release of water “binding” to DNA bp’s such that the hydrophobic ring systems can stack on top of each other to minimize contact with water. Consider the magnitude of stacking energy… Etc.

  29. Forces Stabilizing Biomolecule Structure Proteins Nucleic Acids Hydrophobic Effect Hydrophobic EffectGlobular shape Base Stacking Disulfide Bonds H-Bonds H-bondsAlpha Helices Base Pairing Beta Sheets Ionic Interactions Ionic InteractionsSalt Bridges Metal Ions

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