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Design of a novel globular protein with atomic-level accuracy
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  1. Design of a novel globular protein with atomic-level accuracy

  2. NOVEL METHODS • Computational methods • New developments in the making • Opening of a new field of science

  3. Ideas • Redesign naturally occurring proteins so that they have enhanced stability or new functionality

  4. This group • Found a procedure for the development of low free energy structures • Led them to the creation of the protein TOP7 • A 93 residue a/b protein with topology that is not present in the Protein Structure Data Base

  5. Design Protocol • Critical is cycling between seequence design and backbone optimization. The goal is to find the lowest free energy backbone conformation for a fixed amino acid sequence

  6. Some more Top7 features • Highly soluble protein • Monomeric structure • Thermally stable • More stable than most proteins of its size

  7. Conclusion • Top7 shows that the design of globular proteins not yet observed in nature is possible but can be extremely stable • The methods used to design TOp7 are applicable to any globular protein structure • This may open the door for exploration of new protein strucutres and architectures

  8. A few more signaling things • EGF receptor dimerizes when ligand binds and then causes signaling cascade for cell division uses covalent linkage of phosphate to downstream proteins to carry on signal

  9. Continued • SH molecules (SH2 SH3) are adaptor proteins. • They will recognize specific parts of target molecule and bind and now the phosphate will be transferred on to the target molecule

  10. Calmodulin • Helix loop helix (EF Hand) • Important in many signal transduction pathways • Charged glu and asp bind Ca2+ • Unbound state resembles a dumbbell • Bound state it becomes very compact

  11. Myoglobin/Hemoglobin

  12. Globin Fold • Made up of alpha helices • Hemoglobin and myoglobin are examples

  13. Myoglobin One subunit Transports oxygen in muscle Hemoglobin 4 subunits Transports oxygen all over body Four oxygen binding sites Oxygen is not soluble in blood so it needs carriers

  14. Hemoglobin • Oxygen binds to heme group • No oxygen bound called T state • Oxygen bound called R

  15. Binding of Oxygen • When shifts from T to R the iron moves to the center of the heme plane because an overall structural shift. This shift then gets propagated throughout the structure and leads to the change in other subunits COOPERATIVE BINDING: with the binding of the first The others will bind much easier

  16. Sickle Cell • Base pair change in hemoglobin from glu to val Makes hemoglobin sticky and red blood cells will clump together and cause all sorts of problems

  17. Enzymes • Enzymes speeds things up substantially

  18. Enzymes • Have high specificity for their substrate

  19. How they speed things up • Increase substrate at catalytic site • Physically bring molecules to the right place and increase local concentration • Selectively stabalize by binding to TRANSITION STATE • Lower the activation energy by stabalizing the transition state

  20. ACTIVE SITE • Pocket or groove lined with specific amino acid residues • Here is where the substrate goes and undergoes some sort of chemical transformation • Therefore it can be seen that the enzyme is playing the role in this chemical modification

  21. Serine Proteases

  22. 4 aspects for serine proteases • Catalytic Triad • Oxyanion hole • Specific binding • Non-specific binding

  23. Catalytic Triad Always see these three particular side chains: ASP 102, His 57, and Ser 195 All spread out in primary sequence Come together at active site in binding pocket

  24. Continued His residue accepts proton from reactive serine and helps stabalize the transition state Ser forms covalent bond with substrate

  25. Oxyanion hole Very important in stabalizing the transition state by Forming H Bonds Allows for nonspecific substrate binding to the main chain of the substrate This is the region of the enzyme that is non specific Specific parts are taken care of with the specificity pocket

  26. Specificity binding pocket Accepts different side chains from different substrates depending On the member of the serine protease family

  27. Specificity Pocket

  28. Specificity Pocket Chymotrypsin cleaves bulky aromatic side chains: Serine at the Bottom of the pocket which wont interfere too much with large Aromatic side chain Trypsin cleaves next to large positively charged side chains; Asp At bottom which will attract positive side chain from substrate Elastase cleaves small uncharged sidechains:

  29. Two Phase Reaction First: acylation in which the peptide bond gets cleaved When the substrate comes close, the oxygen of serine 195 Bends to interact with substrate RESULTS in bond between the reactive serine And carboxly or substrate Proton of serine is donated to His Peptide bond goes from planar to tetrahedral state This state is unstable and very short lived

  30. First Phase

  31. Continued This then allows for peptide to be released and the end of the first step

  32. Phase 2 Enzyme still attached to remaining part of peptide Deacylation: water comes along and forms a new tetrahedral Transition state which is also unstable A covalent bond is formed between C and O and the fragment is released Hydrgoen then goes back to serine and enzyme can be used again

  33. Second phase