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Insights into Protein Biosynthesis

Senior Seminar. Department of Chemistry. Insights into Protein Biosynthesis. Entropic Origin of Catalytic Power?. Luigi J. Alvarado Biochemistry, B.S. Class of 2009. April 2, 2009. Outline. Protein Translation The Ribosome Chemistry of Peptide-bond Formation Entropic Phenomenon?

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Insights into Protein Biosynthesis

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  1. Senior Seminar Department of Chemistry Insights into Protein Biosynthesis Entropic Origin of Catalytic Power? Luigi J. Alvarado Biochemistry, B.S. Class of 2009 April 2, 2009

  2. Outline • Protein Translation • The Ribosome • Chemistry of Peptide-bond Formation • Entropic Phenomenon? • Other factors • Conclusions • Aknowledgements

  3. http://www.youtube.com/watch?v=Jml8CFBWcDs&feature=related http://www.youtube.com/watch?v=5bLEDd-PSTQ I. Protein Translation

  4. II. Ribosome • 2.5 MDa/ 4 MDa • rRNA + Protein • Subunits, Domains • A-, P-, E-sites • Tunnels • Peptidyl Transfer Center (PTC)

  5. II. Ribosome (cont’d) - PTC • Domain V of 23S rRNA • No proteins within 15Å • Provides suitable environment • tRNA-binding NTs: • G2251, G2252, A2448, A2450, G2455, U2506, G2583, U2585, and A2602. • 2-fold symmetry • Synchronized rotation duet (45°  180°) • Geometry between the moieties • Solvent reorganization Bashan et al. Mol. Cell2003, 11, 91-102.

  6. II. Ribosome (cont’d) - PTC Bashan et al. Mol. Cell2003, 11, 91-102.

  7. III. Chemistry of Peptide-Bond Formation Ribosomal Reference Six-member Transition Intermediate

  8. III. Chemistry of Peptide-Bond Formation: Ribosomal Rodnina et al. Biochem. Soc. Trans.2005, 33, 493-498.

  9. III. Chemistry of Peptide-Bond Formation: Ribosomal • Nucleophilic attack • Free-tRNA + pept-tRNA • Regio- and stereo-specificity • Methods: Quench-flow assays • 1967: Fragment Rxns  N-blocked aminoacylated oligoNT (CCA-fMet) and Pmn • 2002: pept-tRNA + CPmn Sievers et al. Proc. Natl. Acad. Sci.2004, 101, 7897-7901.

  10. III. Chemistry of Peptide-Bond Formation: Reference • Ester aminolysis • Methods: 1H-NMR • Pseudo-first and second order kinetics • Exclusive attack of the conjugate base of glycinamide. • Polar solvent (+) • Ionic strength (0) Schroeder, G; Wolfenden, R. Biochemistry2007, 46, 4037-4044

  11. III. Chemistry of Peptide-Bond Formation: Reference N-fPhe-TFE Schroeder, G; Wolfenden, R. Biochemistry2007, 46, 4037-4044 N-fPhe-glycinamide N-fPhe

  12. III. Chemistry of Peptide-Bond Formation Six-Member Transition Intermediate Weinger, J.; Strobel, S. Biochemistry2006, 45, 5939-5948.

  13. III. Chemistry of Peptide-Bond Formation Kinetics and Thermodynamics • ΔH‡ = Ea – RT • ΔG‡ = -RTln[(kcat/(KMh))/(kB*T)] • ΔG‡ = ΔH‡ - TΔS‡ Sievers et al. Proc. Natl. Acad. Sci.2004, 101, 7897-7901

  14. III. Chemistry of Peptide-Bond Formation Kinetics and Thermodynamics Kcal/mol M-1s-1 T = 25°C pH = 7.5 Schroeder, G; Wolfenden, R. Biochemistry2007, 46, 4037-4044

  15. Rate Enhancement Ribosomal Reference 1x103 / 3x10-5 = 3x107

  16. IV. Entropic Phenomenon? • ΔΔG‡ ~ -9 kcal/mol • ΔΔH‡ ~8 kcal/mol • ΔTΔS‡ ~18 kcal/mol • What increases the TΔS‡? • Juxtaposition of substrates • Desolvation of PTC • Methods: • MD/EVB simulations • Langevin Dipole solvent, COSMO, and Restraint Release • Explanations: • Proton shuttle model • H-bond network

  17. IV. Entropic Phenomenon? Juxtaposition of the Substrates Beringer, M.; Rodnina, M. Mol. Cell2007, 26, 311-321

  18. IV. Entropic Phenomenon? Desolvation of the PTC Bring reactants to same solvent cage Solvation Orientational Sharma et al. Biochemistry2005, 44, 11307-11314

  19. V. Other Factors H-bond Networks Pre-set Electr. Environ. Beringer, M.; Rodnina, M. Mol. Cell2007, 26, 311-321

  20. VI. Conclusions Schroeder, G; Wolfenden, R. Biochemistry2007, 46, 4037-4044

  21. VI. Conclusions • The Ribosome is an entropy trap • Mechanism of catalysis is not driven via ΔH‡ • Ribosome provides perfect environment • 6-member TI ↔ Proton Shuttle mech. • Other factors’ influence

  22. Aknowledgements • Dr. I. Kovach • Department of Chemistry Faculty • Class of 2009 – 2010 – 2011 • Various researchers

  23. I Chemistry

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