THE WORLD OF THE “NEVER BORN PROTEINS”

1. THE WORLD OF THE “NEVER BORN PROTEINS” by Cristiano Chiarabelli

2. Lightnings_SV3.avi

6. THEORETICAL CONTEXT The proteins existing on our Earth are only an infinitesimal fraction of the theoretically possible ones (Xia & Levitt, 2004). Possible-protein space Natural proteins Determinist theory: The life constituents are the result of an evolutive fine work; what we see is the better possible solution for the biological needs (de Duve, 1995). Contingency theory: extant proteins are the result of the simultaneous interplay of several concomitant causes(Gould, 1994). There may be an entire universe of “Never Born Proteins” (NBP), whose properties have never been sampled by Nature

7. SOME BASIC CALCULATIONS WITH A 50 AMINO ACID PEPTIDE . . . . • 1.1 X 1065 POSSIBLE AMINO ACID COMBINATIONS • SYNTHESIS OF ONE MOLECULE OF EACH COMPOUND: • 1.1 X 1042 KG OF MATERIAL • 1.8 X 1017 TIMES THE WEIGHT OF THE EARTH

8. IT SEEMS UNLIKELY THAT NATURE TRIED ALL COMBINATIONS • IT IS POSSIBLE TO SAMPLE AGAIN AND ISOLATE FOLDED PEPTIDES WITH NO HOMOLOGY TO RECENT PROTEINS Selection

9. AIM OF THE PROJECT FOLDING FREQUENCY DETERMINATION OF RANDOM 50aa POLYPEPTIDES PRESENTING NO HOMOLOGIES WITH EXTANT PROTEINS STRUCTURAL PROPERTIES CHARACTERIZATION

10. mAB 9E10 c-myc TAG P R G Thrombin cleavage site Random Peptide (50mer) ssDNA pIII pVIII Schematic drawing of the filamentous Phage M13 STRATEGY FOR THE ISOLATION OF FOLDED PEPTIDES: PHAGE DISPLAY • The filamentous phage M13 is used in phage display • In A phage the DNA information is “linked“ to the protein • The single-stranded DNA genome is packaged in a long cylinder • M13 is able to infect E. coli • Display of foreign peptides or proteins on pIII • Used in affinity maturation of peptide and protein ligands • Sensitive assay: selection of single phage particles

11. Random fusion protein gene Helper Phage (M13K07) p III C-myc Gene III II X V VII IX M13K07 ColE1 IV Ori M13 Phagemid vector I VI III VIII Ampr ss DNA g III PHAGE DISPLAY TECHNIQUE[1] Phagemid Random fusion protein p III Phagemid DNA Fusion random gene g III [1] Smith, G.P. Science. 1985

12. VALIDATION OF OUR TEST SYSTEM WAS DONE WITH APP APP (Avian Pancreas Peptide - Hormone) protein • 39 aa • NO DISULFIDE BRIDGES • FOLDED • STRUCTURE KNOWN

13. STRUCTURE OF AVIAN PANCREAS PEPTIDE (APP) a-helics Turn Proline helics (PXXP)

14. DESIGN OF THE PHAGEMID VPRG PRG RG No linker APP c-myc tag ColEI Export signal gene III Ap Ori M13

15. THE RESTRICTION SITES VPRG, PRG, RG APP TAG VPRG PRG RG Short flexible linker mAB

16. THROMBIN ASSAY • Phages were incubated 18 hours with • 0.0 U/ml Thrombin • 0.15 U/ml Thrombin • 1.5 U/ml Thrombin • Binding assay enrichment (ELISA) • Samples were used to infect E. coli • Colonies were counted and frequencies were calculated

17. DIFFERENT THROMBIN CLEAVAGE SITES INSERTED UPSTREAM OF APP % digestion by Thrombin 0.0 U/ml 0.15 U/ml 1.5 U/ml

18. CONCLUSIONS • Phage with no cleavage site for Thrombin is not affected by incubation with the enzyme • The sequence RG is not a good Thrombin Substrate • VPRG and PRG are equally good Thrombin Substrate THE PROTEASE CLEAVAGE SITE OF CHOICE IS: PRG

19. NEXT IMPORTANT STEP . . . • proof that Thrombin does not cleave the PRG sequence in a folded peptide

20. THE RESTRICTION SITE PRGPSQ APP P R G TAG short linker mAB

21. THE RESTRICTION SITE PRGPTY APP Turn P R G TAG short linker mAB

22. PRG INSERTED AT DIFFERENT POSITIONS IN APP % digestion by Thrombin 0.0 U/ml 0.15 U/ml 1.5 U/ml

23. CONCLUSIONS - THE MUTANT PROTEINS PRGPTY AND PRGPSQ ARE BETTER PROTECTED AGAINST THROMBIN CLEAVAGE THAN PRG - THE PROLINE HELICS OF APP IS NOT DESTROYED BY THE INSERTION OF PRG AT THE TWO POSITIONS A RANDOM FOLDED PROTEIN WITH THE SEQUENCE PRG INSIDE CAN BE SELECTED USING THROMBIN

24. RANDOM LIBRARY CONSTRUCTION Forward Oligonucleotides Revers Oligonucleotides RANDOM SCHEMA NNK N ( A, C, G, T) K (G, T) NotI XhoI XbaI 69 nt 72 nt XhoI (4 × 4 × 2)47 = 3247~ 1070theoretical random sequences NotI XbaI XhoI XhoI UAG UGA UAA C-myc Gene III DNA RANDOM LIBRARY 47 aa random ColE1 Phagemid Vector [1] Amber mutation: TAG Q Ori M13 Electroporation (Electro Ten-blu, amber mutation) Ampr BamHI NcoI Random selection of 180 colonies XhoI XhoI NotI XbaI C-myc Gene III digestion and ligation NcoI BamHI XhoI NotI XbaI Sequencing 79 correct plasmids C-myc Gene III Pro - Arg - Gly • [1]Lang, S. et al.Biochemistry. 2000

25. RANDOMNESS OF PHAGE PEPTIDE LIBRARY AT THE NUCLEIC ACID LEVEL

26. RANDOMNESS OF PEPTIDE PHAGE LIBRARY AT THE AMINO ACID LEVEL % that an AA occurs in the phage library Theoretical Experimental

27.  PRG c-myc PRG THROMBIN c-myc Random polypeptide (47 aa) Random polypeptide (47 aa) pIII pIII c-myc c-myc Anti-c-myc 9E10 PROTEIN FOLDING SELECTION BY THROMBIN Folded peptides are better resistant to hydrolysis then random-coil ones

28. DISTRIBUTION OF THE PEPTIDE LIBRARY WITH RESPECT TO THROMBIN DIGESTION “Stable” “Unstable” % of the73 clones of the phage library % digestion categories

29. H2N Ala Leu Ile Glu Gln Lys Ser Asp Leu Asn Gly Ala Ala Glu Glu Ala Arg Val c-myc Glu Cys thrombin Asn Met Asp Arg Cys Ile Trp Thr Asp Gly Leu Met Trp Pro Ala Gly His Arg Trp Val Arg Gly Cys O Cys C Cys Leu Lys Ile Leu His Gln Thr Asp Gln Thr Met Leu Thr Ala Ser Thr Gln His His Glu Arg OH Non polar aa – 39% Charged aa – 21% Polar aa – 40% POLYPEPTIDE 127 PRIMARY SEQUENCE

30. native native + urea (6M) Fluorescence (standardized) Fluorescence (standardized) Wavelength (nm) Wavelength (nm) FLUORESCENCE STUDIES Trp λem = 351 127 λem = 343 127 λem (urea 6M) = 343 One or more Tryptophan residues are localized in a zone less exposed to the solvent

31. CD STUDIES CD specta of proteins NBP127. Continue lines for native proteins; dashed lines for proteins at 60 °C; empty triangles for proteins in 6 M urea; empty circles for proteins refolded after 12h dialysis.

33. H2N Ala Leu Ile Glu Gln Lys Ser Asp Leu Asn Gly Ala Ala Glu Glu Ala Met Cys c-myc Cys Arg thrombin Cys Gly Ile Ser Pro Leu Gly Gln Met Gly Tyr Ala Pro Pro Val Trp Gly Glu Gly Ser Arg Gly Ala O Arg C Arg Val Asp Gln Phe Pro Met Thr Arg Asn Thr Gln Leu Thr Ser Leu His Val Ala Ser Pro Pro OH Non polar aa – 55% Charged aa – 13% Polar aa – 32% POLYPEPTIDE 1 PRIMARY SEQUENCE

34. CD STUDIES CD specta of proteins NBP1. Continue lines for native proteins; dashed lines for proteins at 60 °C; empty triangles for proteins in 6 M urea; empty circles for proteins refolded after 12h dialysis.

35. : no polar zones : polar zones : + residues : - residues TRIDIMENTIONAL STRUCTURE PREDICTION • Globular Structure: • 3 α-Helices: • Res. 9 → 22 • Res. 27 →30 • Res. 42 → 52 (PRG) Tot. α-Helix ~ 40%

36. C-terminal TRIDIMENTIONAL STRUCTURE PREDICTION Cys residues

37. Trp 23 C-terminal N-terminal FLUORESCENCE STUDIES Fluorescence (standardized) Wavelength (nm) • The Trp residue could be exposed to the solvent

38. HIGHLIGHTS • A PHAGE DISPLAY METHOD FOR THE ISOLATION OF THROMBIN-RESISTANT PEPTIDES WAS DEVELOPED • MAJORITY OF THE CLONES ARE EITHER FULLY RESISTANT AGAINST THROMBIN OR COMPLETELY DIGESTED BY THROMBIN • HIGH PERCENTAGE OF THROMBIN-RESISTANT PEPTIDES • HIGH PERCENTAGE OF FOLDED PEPTIDES IN A RANDOM-PEPTIDE POPULATION

39. OUTLOOK • STRUCTURAL STUDIES OF MORE THROMBIN-RESISTANT PEPTIDES • -cd measurements • -nmr • -crystals • COMPUTATIONAL ANALYSIS OF THE PEPTIDE SEQUENCES • -codon statistics • -positional scanning • -nearest-neighbor analysis • -comparison with E. coli and Human small proteins • ISOLATION OF FOLDED RNA FROM THE PHAGE LIBRARY • Insertion of T7 promoter in the current library • Selection with RNAse to select folded RNA