Peptide+structure

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Peptide+structure

  1. 1. PEPTIDE STRUCTURE - FUNCTION
  2. 2. Rational Design of Peptides - Driving Force CRYSTALLOGRAPHY NMR – HIGH RES ABS, FLUORES, CD, IR - LOW SEQUENCING SEQUENCE STRUCTURE
  3. 3. + PEPTIDE BOND FORMATION AA1 AA2 H 2 0 DIPEPTIDE PROTEASES H 2 NC  HC OH O R HHNC  HC OH O R H 2 NC  HC O R 1 HNC  HC OH O R 2
  4. 4. PHYSICO-CHEMICAL PROPERTIES PHYSICAL PROPERTIES ADDITIVE LEGNTH, MASS NOT ADDI IVE Pka => AA1+AA2 ===== DIPEPEPTIDE ENERGETICS, REACTIVITY ETC
  5. 5. STRUCTURE OF THE PEPTIDE BOND
  6. 6. GEOMETRICAL CONSTRAINTS - CONFORMATIONS ALLOWED NOT-ALLOWED ANGLES
  7. 7. DIPOLE ORIGIN OF PEPTIDE BOND PLANE NOT ALL CONFORMATIONS POSSIBLE PREFERED CONFORMATIONS
  8. 8. STRUCTURAL MOTIFS - FUNCTIONAL <ul><li>HELIX -  -HELIX, 3-10 HELIX </li></ul><ul><li> -SHEETS (Parallel, Anti-Parallel </li></ul><ul><li> -TURNS </li></ul><ul><li>RANDOM COILS </li></ul>
  9. 9.  helix <ul><li>α-helix (30-35%) </li></ul><ul><ul><li>Hydrogen bond between C=O (carbonyl) & NH (amine) groups within strand (4 positions apart) </li></ul></ul><ul><ul><li>3.6 residues / turn, 1.5 Å rise / residue </li></ul></ul><ul><ul><li>Typically right hand turn </li></ul></ul><ul><ul><li>Most abundant secondary structure </li></ul></ul><ul><ul><li>α-helix formers: A,R,L,M,E,Q,K </li></ul></ul>
  10. 10. the alpha-helix: repeating i,i+4 h-bonds 2 1 3 4 5 7 8 9 6 10 11 12 right-handed helical region of phi-psi space hydrogen bond
  11. 11. The  -helix, with i,i+4 h-bonds, is not the only way to have local hydrogen bonding of the backbone to itself. The 3 10 helix has hydrogen bonds between residues i and i+3 The  helix has hydrogen bonds between residues i and i+5. For a number of reasons almost all helices in proteins are  -helices--include backbone, side chain steric issues, van der Waals contacts, H-bond geometry  -helix 3 10 helix  helix these are poly-Ala, so the gray balls on the outside are  -carbons from the side chains
  12. 12.  sheet &  turn <ul><li>β-sheet / β-strand (20-25%) </li></ul><ul><ul><li>Hydrogen bond between groups across strands </li></ul></ul><ul><ul><li>Forms parallel and antiparallel pleated sheets </li></ul></ul><ul><ul><li>Amino acids less compact – 3.5 Å between adjacent residues </li></ul></ul><ul><ul><li>Residues alternate above and </li></ul></ul><ul><ul><li>below β-sheet </li></ul></ul><ul><ul><li>β-sheet formers: V,I,P,T,W </li></ul></ul>
  13. 14. <ul><li>β-turn </li></ul><ul><ul><li>Short turn (4 residues) </li></ul></ul><ul><ul><li>Hydrogen bond between C=O & </li></ul></ul><ul><ul><li>NH groups within strand </li></ul></ul><ul><ul><li>(3 positions apart) </li></ul></ul><ul><ul><li>Usually polar, found near surface </li></ul></ul><ul><ul><li>β-turn formers: S,D,N,P,R </li></ul></ul>TURNS
  14. 15. Others <ul><li>Loop (bridging region) </li></ul><ul><ul><li>Regions between α-helices and β-sheets </li></ul></ul><ul><ul><li>On the surface, vary in length and 3D configurations </li></ul></ul><ul><ul><li>Do not have regular periodic structures </li></ul></ul><ul><ul><li>Loop formers: small polar residues </li></ul></ul><ul><li>Coil (40-50%) </li></ul><ul><ul><li>Generally speaking, anything besides α-helix, β-sheet, β-turn </li></ul></ul>
  15. 16. Principal types of secondary structure found in proteins Repeating (  ) values -63 o -42 o -57 o -30 o -119 o +113 o -139 o +135 o   -helix (1  5) (right-handed)    helix (1  4) Parallel  -sheet Antiparallel  -sheet
  16. 17. STRUCTURES IN ACTION GCN4 “leucine zipper” (green) bound as a dimer (two copies of the polypeptide) to target DNA The GCN4 dimer is formed through hydrophobic interactions between leucines (red) in the two polypeptide chains Leu Leu
  17. 18. TECHNIQUES – PEPTIDE COMFORMATION CD X-ray Crystallography NMR
  18. 19. Do Small Peptides have Conformation Yes & No. S-Peptide Ribonuclease A – Helical structure in solution Use of Helix Inducing Solvents – TFE and N-Propanol
  19. 20. Helix Induction and Propensity
  20. 21. CONFORMATIONAL TRANSITION
  21. 23. PROPENSITY CALCULATION
  22. 24. BIOLOGY OF PEPTIDES RIBOSOMAL PROTEINS NON-RIBOSOMAL PEPTIDES SPECIFIC ENZYMES PROTEOLYTICALY PROCESSED DEGRADED TO AA (Antibiotics, phytochelatins) (Enzymes) MHC Peptides
  23. 25. CHEMICAL METHOD –PEPTIDE SYNTHESIS STAGE 1: ASSEMBLE AA ON POLYMER SUPPORT (R – PROTECTED) NON-REACTIVE STAGE 2: CLEAVE THE SYNTHESIZED PEPTIDE a) CLEAVAGE OF CHAIN b) DE-PROTECT SIDE CHAIN STAGE 3: PURIFY CRUDE PEPTIDES – HPLC STAGE 4: STORAGE – LYOPHILIZE, SPEEDVAC, ETC STAGE 5: SEQUENCE, MALDI-TOF
  24. 26. SOLID-PHASE PEPTIDE SYNTHESIS (SPPS) STAGE 1: a) Attach N-terminal + Side Chain Protected to Polymer Support (Activation of C & Coupling to Support) b) Deprotection (N-term ) c) Coupling Next AA (Protected) d) Deprotection (N-term) Continued ….
  25. 27. V 8 Protease “ Conformational Trap” Protease-mediated Protein Splicing – Nature’s Choice LYGSTSQE VASVKQAFDAVGVK NH-VASVKQAFDAVGVK-OH NH-LYGSTSQE-OH “ Proteolysis” “ Reverse Proteolysis” LYGSTSQE VASVKQAFDAVGVK
  26. 28. TAAAKFE “ Conformational Trap” can act alone
  27. 29. Conformational Trap of product – ambient conditions, easy Isolation, and Purification of Products Applications 1. Ability to Incorporate Non-Natural aminoacids or synthesize Man-made peptides or proteins of therapeutic interest Semisynthetic Insulin, Hemoglobin, and IL-10 Sortases – Glycoprotein synthesis Laboratory reagents :- Protein with reporter groups, Kinases or Phosphotases with Pmp(phosphonomethylene phenylalanine )

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