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Peptide+structure

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  • 1. PEPTIDE STRUCTURE - FUNCTION
  • 2. Rational Design of Peptides - Driving Force CRYSTALLOGRAPHY NMR – HIGH RES ABS, FLUORES, CD, IR - LOW SEQUENCING SEQUENCE STRUCTURE
  • 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. PHYSICO-CHEMICAL PROPERTIES PHYSICAL PROPERTIES ADDITIVE LEGNTH, MASS NOT ADDI IVE Pka => AA1+AA2 ===== DIPEPEPTIDE ENERGETICS, REACTIVITY ETC
  • 5. STRUCTURE OF THE PEPTIDE BOND
  • 6. GEOMETRICAL CONSTRAINTS - CONFORMATIONS ALLOWED NOT-ALLOWED ANGLES
  • 7. DIPOLE ORIGIN OF PEPTIDE BOND PLANE NOT ALL CONFORMATIONS POSSIBLE PREFERED CONFORMATIONS
  • 8. STRUCTURAL MOTIFS - FUNCTIONAL
    • HELIX -  -HELIX, 3-10 HELIX
    •  -SHEETS (Parallel, Anti-Parallel
    •  -TURNS
    • RANDOM COILS
  • 9.  helix
    • α-helix (30-35%)
      • Hydrogen bond between C=O (carbonyl) & NH (amine) groups within strand (4 positions apart)
      • 3.6 residues / turn, 1.5 Å rise / residue
      • Typically right hand turn
      • Most abundant secondary structure
      • α-helix formers: A,R,L,M,E,Q,K
  • 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. 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.  sheet &  turn
    • β-sheet / β-strand (20-25%)
      • Hydrogen bond between groups across strands
      • Forms parallel and antiparallel pleated sheets
      • Amino acids less compact – 3.5 Å between adjacent residues
      • Residues alternate above and
      • below β-sheet
      • β-sheet formers: V,I,P,T,W
  • 13.  
  • 14.
    • β-turn
      • Short turn (4 residues)
      • Hydrogen bond between C=O &
      • NH groups within strand
      • (3 positions apart)
      • Usually polar, found near surface
      • β-turn formers: S,D,N,P,R
    TURNS
  • 15. Others
    • Loop (bridging region)
      • Regions between α-helices and β-sheets
      • On the surface, vary in length and 3D configurations
      • Do not have regular periodic structures
      • Loop formers: small polar residues
    • Coil (40-50%)
      • Generally speaking, anything besides α-helix, β-sheet, β-turn
  • 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
  • 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
  • 18. TECHNIQUES – PEPTIDE COMFORMATION CD X-ray Crystallography NMR
  • 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
  • 20. Helix Induction and Propensity
  • 21. CONFORMATIONAL TRANSITION
  • 22.  
  • 23. PROPENSITY CALCULATION
  • 24. BIOLOGY OF PEPTIDES RIBOSOMAL PROTEINS NON-RIBOSOMAL PEPTIDES SPECIFIC ENZYMES PROTEOLYTICALY PROCESSED DEGRADED TO AA (Antibiotics, phytochelatins) (Enzymes) MHC Peptides
  • 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
  • 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 ….
  • 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
  • 28. TAAAKFE “ Conformational Trap” can act alone
  • 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 )