1. Self-Organizing Bio- structures NB2-2009L.Duroux

2. Lecture 2 Macromolecular Sequences

3. Introduction-questions: How do we move along from prebiotic small molecules to oligomers and polymers (DNA & proteins)? How do we move along from prebiotic small molecules to oligomers and polymers (DNA & proteins)? Why the need for long polymeric chains vs cooperation of small ones? Why the need for long polymeric chains vs cooperation of small ones? Why are proteins long polypeptides? Why are proteins long polypeptides?

4. What is the easiest way to get a functional bio-catalyst? Lysozyme

5. Examples of the ”necessity” for growing larger peptides Protein domains

6. A common case of ”chain-growth”: Protein structural domains Chymotrypsin ‘Modern’ 2-  -barrel structure Putative ancestral  -barrel structure Active site (combination of ancestral active site residues) Activity 1000-10,000 times enhanced

7. 3D structure of Chymotrypsin

8.  barrel regulatory domain  barrel catalytic substrate binding domain  nucleotide binding domain 1 continuous + 2 discontinuous domains A multiple-domain protein: pyruvate kinase

9. Co-polymerization A step towards macromolecules

10. Famous natural copolymers

11. Model for a copolymer growth r A = k AA / k AB and r B = k BB / k BA

12. Copolymer composition as function of r A and r B Modelized by Mayo-Lewis equation Modelized by Mayo-Lewis equation r A = r B >> 1 : homopolymers (AAAA or BBBB) r A = r B >> 1 : homopolymers (AAAA or BBBB) r A = r B > 1 : block-copolymer (AAAAABBBBBB) r A = r B > 1 : block-copolymer (AAAAABBBBBB) r A = r B ≈ 1 : random copolymer (AABAAABBABBB) r A = r B ≈ 1 : random copolymer (AABAAABBABBB) r A = r B ≈ 0 : alternate copolymer (ABABABABABA) r A = r B ≈ 0 : alternate copolymer (ABABABABABA) Example: Example: Maleic anhydride ( r A = 0.03) Maleic anhydride ( r A = 0.03) trans-stilbene ( r B = 0.03) trans-stilbene ( r B = 0.03)

13. Monomer Addition by Radical propagation radical attacks double bond of monomer radical attacks double bond of monomer new radical forms that is one monomer unit longer new radical forms that is one monomer unit longer chain reaction chain reaction The polymer chain grows by addition of monomer units: The polymer chain grows by addition of monomer units: chain has propagated chain has propagated called free radical polymerisation called free radical polymerisation

14. Rubber : a natural case of addition (co)polymerization

15. Radical Initiation Q:From where does the first unpaired electron come? Q:From where does the first unpaired electron come? A:Generated by an initiator A:Generated by an initiator e.g. hydrogen peroxide (H 2 O 2 ) e.g. hydrogen peroxide (H 2 O 2 ) has O–O bond (easy to break) has O–O bond (easy to break) generates 2 OH radicals generates 2 OH radicals usually don’t use H 2 O 2 but other peroxides, e.g.: usually don’t use H 2 O 2 but other peroxides, e.g.: potassium persulfate potassium persulfate persulfate ion is: [O 3 S–O–O–SO 3 ] 2– persulfate ion is: [O 3 S–O–O–SO 3 ] 2– O–O bond breaks readily at 60 o C to initiate reaction O–O bond breaks readily at 60 o C to initiate reaction

16. Some Common Polymers polyethylene (also called polythene) polyethylene (also called polythene) Glad Wrap poly(vinyl acetate) (PVAc) poly(vinyl acetate) (PVAc) glues, paints poly(vinyl alcohol) (PVA) poly(vinyl alcohol) (PVA)glues polystyrene polystyrene bean bags, packing

17. Polypeptides, polynucleotides: more difficult! Chain composition difficult to predict: Chain composition difficult to predict: Several co-monomers (20 aa, 5nt) Several co-monomers (20 aa, 5nt) Monomer concentrations might vary Monomer concentrations might vary Complex interplay between many kinetic parameters Complex interplay between many kinetic parameters Condensation polymerization (≠ addition) Condensation polymerization (≠ addition) Thermodynamics not favorable Thermodynamics not favorable Needs activation (energy) Needs activation (energy)

18. Prebiotic activation of monomers

19. Formation of homo-polypeptides H2O a problem ! Condensation possible on clay AMP not a pre-biotic molecule!

20. Other routes to condensation of amino-acids From amino-acids: From amino-acids: Possible in vesicles without activation + heat Possible in vesicles without activation + heat Heat 180˚C + excess Glu/Asp or Lys Heat 180˚C + excess Glu/Asp or Lys Metal ions + Drying + Heat Metal ions + Drying + Heat Condensation Condensation HCN + addition of side chains HCN + addition of side chains N-carboxyanhydrides (see Chap. 3) N-carboxyanhydrides (see Chap. 3) Carbonyl sulfide: COS (prebiotic volcanic gas) Carbonyl sulfide: COS (prebiotic volcanic gas) Questions: Questions: What about chains longer than 10 amino-acids? What about chains longer than 10 amino-acids? What about chain sequence specificity? What about chain sequence specificity?

21. The case of polynucleotides Activated nucleotide: Phosphorimidazolide (b) stereospecificity 3’-5’ (c) Clay: water activity reduced UV-resistance

22. Template-directed oligomerization Still : No explanation for NMPs No explanation for the retention of particular sequences of nucleotides

23. The problem of peptide chains ”selection” & never-born proteins...

24. Aetiology of the current protein set Consider a chain of 100aa : 20 100 possibilities! Consider a chain of 100aa : 20 100 possibilities! Total number of natural proteins: 10 15 Total number of natural proteins: 10 15 Now: 10 15 / 20 100 ≈ r H / r universe Now: 10 15 / 20 100 ≈ r H / r universe What about the ”never-born” or ”obliterated” proteins? What about the ”never-born” or ”obliterated” proteins? Only one reasonable assumption to limit the set: contingency + thermodynamics! Only one reasonable assumption to limit the set: contingency + thermodynamics!

25. The ”never-born” or ”obliterated” proteins: do they fold? Is there anything special about the proteins we know (energy, folding...)? Is there anything special about the proteins we know (energy, folding...)? Experimental test: Experimental test: Screening random-generated peptide library (50aa) Screening random-generated peptide library (50aa) Do they fold? Do they fold?

26. Never-Born proteins: experimental set-up Only folded peptides resist to thrombin cleavage 80 clones tested: 20% resistant

27. The problem of formation (and ”selection”) of macromolecular sequences

28. In which conditions? Oligopeptides formed (up to 10aa) in various libraries, in prebiotic conditions Oligopeptides formed (up to 10aa) in various libraries, in prebiotic conditions Condensation of oligopeptides possible: Condensation of oligopeptides possible: Catalytic dipeptides (seryl-histidine, histidyl-histidine) Catalytic dipeptides (seryl-histidine, histidyl-histidine) Reverse reaction favoured in H2O-free medium Reverse reaction favoured in H2O-free medium Clay support or phase-separation (product insoluble) Clay support or phase-separation (product insoluble)

29. Peptide-fragments condensation As a result of contingency: pH, salinity, temperature... * Catalytic residue = peptidase activity specific to terminal amino acid

30. A double, independent origin of macromolecules? And life could begin...?

31. Homochirality in chains & chain growth

32. Synthetic Homochirality The case of vinyl polymers : polypropylene (G. Natta) Confers helical conformations to polymer in crystals

33. Theoretical model for chain chirality Enantiomeric excess: (D-L)/(D+L) = 0.2 => 60% D + 40% L Dn/Ln grows exponentially with n power (binomial distribution) Enantiomeric excess = 1 when n=20! Homo-poly-Leu

34. Relative abundance of homochiral chains of homo-polypeptides (Trp) White: random distribution Grey: observed composition Over-representation of homochiral peptides

35. Conclusions Prebiotic chemistry could explain formation of short peptide chains / oligonucleotides Prebiotic chemistry could explain formation of short peptide chains / oligonucleotides Still problems with activation chemistry Still problems with activation chemistry Copolymerization Rules explain chain composition Copolymerization Rules explain chain composition Never-born proteins universe is huge: some NBP can fold Never-born proteins universe is huge: some NBP can fold Homochirality in chains is naturally selected, can be explained statistically. Homochirality in chains is naturally selected, can be explained statistically.