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Insights into Protein Biosynthesis

Insights into Protein Biosynthesis

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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