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A new approach towards deciphering the protein code: The protein assembly model

A new approach towards deciphering the protein code: The protein assembly model. Claire Lesieur lesieur@lapp.in2p3.fr. Membrane (Lipids). Proteins. Nucleus (chromosome). Elements of the living world. Protein. Nucleus. Lipids. DNA. CHON. Chromosome. Protein Biological activities.

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A new approach towards deciphering the protein code: The protein assembly model

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  1. A new approach towards deciphering the protein code: The protein assembly model Claire Lesieur lesieur@lapp.in2p3.fr

  2. Membrane (Lipids) Proteins Nucleus (chromosome) Elements of the living world Protein Nucleus Lipids DNA CHON Chromosome

  3. Protein Biological activities • Cutting • Recognition • Enzyme • Signaling • Carrier • Shape generator • Road networks

  4. Structure-function relationshipin proteins • Function • Shape • How the shape provides a particular function • How the shape is acquired

  5. ? ? GKKHDGATTYQW

  6. The protein folding problem • How it folds: Mechanisms of protein folding • How the information is encrypted in the sequences: CODING problem ADRTGGILLKMHGGARECVVP

  7. All the information necessary for the protein folding is within the protein primary sequence C.B. Anfinsen, Haber, E., Sela, M. & White, F. H. , Proc. Nati. Acad. Sci. USA 47 (1961) 1309-1314. Levinthal’s paradox(1968): not random search but directed Levinthal, C. (1968) J. Chim. Phys. 65, 44-45.

  8. COOH H2N s-hours ms Structure Tertiaire Structure primaire Structure Secondaire Mechanism Short range interaction long-range interactions short-range interactions

  9. Code: still unknown X-ray crystallography + NMR: PDB 3D modeling: PDB ~ 70 % Sequence similarity: 3D modeling 70 % similarity: different shape Low sequence similarity: similar shape Amino acids on the surface of proteins: changeable

  10. Transmembrane domains of Membrane proteins b-strands transmembrane domain: 1010101 a-helicetransmembrane domain: 11111111111111111

  11. Biologically active amino acids

  12. Sequence-Shape predictions • Geometrical constrain • Chemical constrain

  13. To read sequences you need to determined comparable sequences • Domains • Shape and role ? Sequence Pattern ? Sequence Pattern

  14. Protein assembly

  15. Aerolysine Trends in Microbiology (2000). Vol 8 (4):169-172

  16. ER Cholera toxin CtxA CtxB5 • AB5 toxin • A catalytic subunit • B receptor binding subunit • GM1: cell receptor • Endocytosed and traffic to the ER • ADP ribosylation of Ga subunit • Increase of cAMP leading to water loss

  17. Experimental approach

  18. Assembly in vitro pH 7 pH 1 15 min Pentamere Monomere

  19. 2D structural level: short range interaction 5 2 10 0 5 -2 10 5 -4 10 pH 1 5 -6 10 Mean residue Molecular Ellipticity pH 7 5 -8 10 Native 6 -1 10 6 -1,2 10 200 210 220 230 240 250 Wavelength (nm)

  20. 3D structural level: long range interaction • Trp-fluorescence 300 lex= 295 nm lem=352 nm Fluorescence Intensity (a.u.) 200 Fluorescence Intensity 100 unfolded 0 Time (min) 320 340 360 380 Wavelength (nm)

  21. Functional test His CtxB 100 80 Function 60 HISTIDINE 40 20 4,5 5 5,5 6 6,5 7 7,5 8 0 pH

  22. … CtxB5 …

  23. LTB CtxB Cholera toxin B Heat labile enterotoxin B

  24. N-terminal 100 LTB CtxB 80 Function 60 N-terminal 40 20 0 4,5 5 5,5 6 6,5 7 7,5 8 pH

  25. LTB5

  26. Kinetics differences On pathway intermediates differences It is particular amino acids that are responsible for each individual step of assembly and folding

  27. Fundamental question • Alzheimer, Parkinson, Prion diseases Protein X: FOLD state: healthy Information for interfaces (Protein X)n: Assembly state: Lethal

  28. Theoritical approach • Protein Interface formation • Rules? • Mechanism? • Preferential geometries related to preferential sequences of amino acids?

  29. INTERFACES: Zone de contact entre monomeres voisins

  30. Analyses des interfaces Interface Trimer pentamer heptamer Brin 1 Brin 2 0101 0101 Ch111Ch n.a. Ch111Ch 1111/1

  31. Trimeric Domain

  32. Fibritin like domain

  33. Oligomeric proteins Nombre de monomer 2 3 4 5 6 7 8 9 10 11 12 Nombre de cas 5722 1035 2340 168 721 46 512 45 87 8 205

  34. Programme detection Protein Interfaces Monomer M 513 -524 LMITTECMVTDL aaa-bbbbbbb- Monomer M+ 1 35-49 GRNVVLDKSFGAPTI --bbbb-------bb Distances

  35. 2HY6 (30) 1 30 beta 1N9R (68) 19 86 alpha 1WNR (94) 1 94 a+b 2F86 (129) 344 471 1JBM (78) 10 88 rc 1G31 (107) 5 111 1LNX (74) 8 80 1Q57 (483) 64 549 2RAQ (94) 3 97 1GRL (518) 6 523 1IOK (524) 2 526 1PZN (240) 96 336 1J2P (229) 4 233 1Y7O(194) 1 194 2F6I (189) 177 367 1TG6 (193) 1 193 2CBY (179) 15 194 1OEL (525) 2 525 1LEP (92) 1 92 3BDU (51) 2 53 1HX5 (92) 5 97

  36. PUTATIVE LIPOPROTEIN from E. CAROTOVORA 3BDU 20-29, 38-53

  37. Common protein interfaces of unrelated proteins 3BDU 1--111011-110110--10 1G31 0--1-1001-100100--00 1JBM 11001000101100101101 1LNX 1--0100010110000---1 1N9R 0--0100011110010--11 1J2P ----1000101100101--1 1HX5 ------0011110010--11 1LEP 0---10001000--00--11 Con2 ----1-001-1100-0-

  38. 1LEP: 1-8, 88-94, 40-57 1WNR: 1-8, 88-94, 44-57, 62-77 1HX5: 5-11, 94-97, 51-62, 68-80,27-30 1G31: 8-15, 104-111, 68-85

  39. 1N9R yeast Methanobacterium Thermautriophicum: extremophile 1JBM P. aerophilum: bacterium 1LNX

  40. 1 yeast 1 + 1 Methanobacterium Thermautriophicum: extremophile 1JBM: 12-18, 42-50, 64-83 1 +1 +1 1N9R: 66-82 P. Aerophilum Hyperthermophilic bacterium 1LNX: 10-15, 25-32, 40-48, 63-77

  41. 2CBY

  42. Conclusion • Geometry and function related • Family of protein interfaces • Assembly keys

  43. Future • Classification of protein interfaces: Database • Systematic analysis of protein interfaces-subjective classification • Mathematical approach: Laurent Vuillon (LAMA) • Functional analysis of protein interfaces • Protein Assembly mechanism from block: Giovanni Feverati • Stoechiometry/Symmetry: Paul Sorba • Experimental tests: Claire Lesieur

  44. Acknowledgment • Alicia Ng Ling • Mun Keat Chong • Boon Leng Chua • Danyang Kong • Giovanni Feverati • Paul Sorba

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