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

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

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

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 )