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4D STRUCTURE OF PROTEINS

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4D STRUCTURE OF PROTEINS

  1. 1. Protein 3-D structure: 3o and 4o structure and protein folding.
  2. 2. 3o Structure third level of protein organization folding of polypeptide chain causes 2o structures to interact formation of motifs and domains
  3. 3. Proteins with similar 1o structure also have similar 3o structure tuna 1 GDVAKGKKTFVQKCAQCHTVENGGKHKVGPNLWGLFGRKTGQAEGYSYTDANKSKGIVWN yeast 1 GSAKKGATLFKTRCLQCHTVEKGGPHKVGPNLHGIFGRHSGQAEGYSYTDANIKKNVWDE rice 1 GNPKAGEKIFKTKCAQCHTVDKGAGHKQGPNLNGLFGRQSGTTPGYSYSTANKMAVIWEE tuna 61 ETLMEYLENPKKYIPGTKMIFAGIKKKGERQDLVAYLKSATS yeast 61 NNMSEYLTNPKKYIPGTKMAFGGLKKEKDRNDLITYLKKACE rice 61 NTLYDYLLNPKKYIPGTKMVFPGLKKPQERADLISYLKEATS
  4. 4. Common Motifs
  5. 5. Motifs Combine to form Domains Domains are independent folding units in a 3o structure of a protein Individual domains have specific function Hydrophobic interactions are the major driving force in folding domains Parallel twisted sheet Alpha/beta barrel
  6. 6. Protein family members share common domain structures lactate dehydrogenase malate dehydrogenase COO- COOO C + NADH CH3 COOHO CH + NAD+ CH 3 O C H2C + NADH COO- COOHO CH + NAD+ CH2 COO-
  7. 7. 4 o Structure •Quaternary structure describes the organization of subunits in a protein with multiple subunits (oligomeric protein) •Can have homo-multimers or heteromultimers
  8. 8. a2b2 a2bg
  9. 9. 4 o Structure Determine molecular weight of native protein by gel permeation chromatography Determine molecular weight of individual subunits by SDS-PAGE Can use the information to determine subunit composition
  10. 10. If……. Native protein – 160,000 daltons and a-Subunit – 50,000 daltons b-Subunit – 30,000 daltons Then…… Protein can have a2b2 structure
  11. 11. 4 o Structure Subunits held together by non-covalent interactions Oligomeric protein is more stable than disassociated subunits Active site often made up of AA residues from different subunits 4o and 3o structure is often affected by ligand (substrate or inhibitor) binding. Important in enzyme regulation
  12. 12. Protein denaturation Denaturation – disruption of native conformation Heat commonly used to denature proteins Tm = temperature where 50% folded/50% unfolded. Typical Tm = 40-60oC Tm for thermophiles >100oC (Taq DNA polymerase) Chemical denaturants Chaotrophic agents = Urea, KCN detergents = SDS
  13. 13. Protein Folding Ribonuclease A (RNase A) will refold to native structure spontaneously (1 minute) >1050 possible conformations If 10-13 sec per conformation would take 1030 years to sample enough to determine structure How do proteins fold so quickly?
  14. 14. Factors driving protein folding Conformational entropy A+B  C decreases entropy (unfavorable) Non-covalent interactions give favorable enthalpy value Hydrophobic effect increases entropy by freeing water (favorable)
  15. 15. DG = DH - TDS - +
  16. 16. Protein Folding Structures of globular proteins are not static Proteins “breathing” between different conformations Proteins fold towards lowest energy conformation Multiple paths to lowest energy form
  17. 17. All folding paths funnel towards lowest energy form Local low energy minimum can slow progress towards lowest energy form
  18. 18. Pathway of Protein Folding 1) Nucleation of folding Rapid and reversible formation of local 2o structures form
  19. 19. 2) Formation of domains (Molten Globular intermediates) through aggregation of local 2o structures 3) Domain conformations adjust to form native protein
  20. 20. Chaperonins Protein complexes that promote protein folding Chaperonins don’t determine native structure Prevent misfolding and aggregation of protein Sequesters unfolded protein from other proteins
  21. 21. Require ATP for protein binding, after ATP hydrolysis native protein released Thought to bind unfolded regions of protein
  22. 22. Disulfides Bonds Stabilize native structure Formed after native conformation achieved Abundant in secreted proteins but not in intracellular proteins Protein disulfide isomerase catalyzes reduction of incorrect disulfide linkages

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