1. Conformational Analysis of a Set of Peptides Corresponding to the Entire Primary Sequence of the N-terminal Domain of the Ribosomal Protein L9: Evidence for Stable Native-like Secondary Structure in the Unfolded State Donna L. Luisi, Wen-Jin Wu and Daniel P. Raleigh* J. Mol. Biol. (1999) 287, 395-407. Speaker:Wu Chih-Wei Date : 2000/3/24

2. Introduction to Ribosome  Ribosome contains one mRNA and two tRNA binding sites  It contains two subunits 50S and 30S

3. Structure of Ribosome Molecular biology of the cell. P.232

4. Schematic localization of L9 protein on the 50S ribosomal subunit J.B.C. (1991) 266..33. 22129-22135.

5. The structure of L9 protein

6. Why Protein L9?  L9 forms an interesting bilobal structure with a compact N-terminal domain connected by a long solvent-exposed α -helix to a compact C-terminal domain.  Protein L9 does not appear to participate in subunit interaction nor in peptidyltransferase activity.  L9 is one of the simplest examples of sheet-helix structures.  Lack disulfide bond and no cofactors

7. N-terminal domain of L9

8. CD spectra of the five peptides PH 5.4, 4℃ β1:1-11 β2:12-23 α1: 24-34 β3:35-42 α2:40-56 206nm 222nm

9. CD standard curve α: αhelix β : β sheet Rc : random coil

10. Far-UV CD spectra of α-1 □ : 75 μ M ● : 500 μ M Below 100μM random coil 100-545 μM not random coil, (self-assocites)

11. NMR spectrum of β-2 DQF-COSY spectrumROESY spectrum The β-1, β-2, β-3 peptide are unstructure in solution.

12. Summary of NMR data CSI : +1 (βsheet) 0 (no structure) –1 (α-helix) 3 J HNCα : ● : below 6 HZ (α-helix) ○ : above 8 HZ(β-sheet) － : not measure : 6-8 HZ(random coli)

13. The α-2 Peptide NMR Spectrum PH 5.4, 4℃ DQF-COSY spectrumNOESY spectrum Get 9 amino acid and 2 of 5 are too close to the diagonal. Provide more direct evidence for the helix formation.

14. A native N-capping interaction stabilizes the helical structure α-2 : 40-56 (53% helix) PH 5.4 4℃ α-2B: 41-56 (32% helix )

15. CD spectra of the peptide α-2 and α-2B

16. Conclusion  The pH and ionic strength dependence of the helical content of α-2.  The change in θ 222 from 0.4M to 1.6M NaCl is less than 3%.  The change in θ 222 in PH 11 is less than 10%.  The change in θ 222 in PH 2 is more than 10%, corresponding to an apparent increase in helicity.  Thr40 acts as an N-capping residue and its side-chain forms a H-bond with the amide proton of residue 43.  The peptides β -3 and α -2 provide a model of cis-trans proline isomerism in the unfolded state.  Trans C α Thr40– C δ Pro41 (native state)  Cis C α Thr40– C α Pro41

17. Cis-trans proline isomerism

18. ~ The End