Speaker:Wu Chih-Wei Date : 2000/3/24
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Transcript of Speaker:Wu Chih-Wei Date : 2000/3/24
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 St
ateDonna L. Luisi, Wen-Jin Wu and Daniel P. Raleigh*
J. Mol. Biol. (1999) 287, 395-407.Speaker:Wu Chih-Wei
Date : 2000/3/24
Introduction to Ribosome
Ribosome contains one mRNA and two tRNA binding sites
It contains two subunits 50S and 30S
Structure of Ribosome
Molecular biology of the cell. P.232
Schematic localization of L9 protein on the 50S ribosomal
subunit
J.B.C. (1991) 266. .33. 22129-22135.
The structure of L9 protein
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
N-terminal domain of L9
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
CD standard curve
α: αhelix
β : β sheet
Rc : random coil
Far-UV CD spectra of α-1
□ : 75μM ● : 500 μM
Below 100μM random coil
100-545 μM not random coil, (self-assocites)
NMR spectrum of β-2
DQF-COSY spectrum ROESY spectrum
The β-1, β-2, β-3 peptide are unstructure in solution.
Summary of NMR data
CSI : +1 (βsheet)
0 (no structure)
–1 (α-helix)
3JHNCα:● : below 6 HZ (α-helix)
○ : above 8 HZ(β-sheet)
- : not measure
: 6-8 HZ(random coli)
The α-2 Peptide NMR SpectrumPH 5.4 , 4℃
DQF-COSY spectrum NOESY spectrum
Get 9 amino acid and 2 of 5 are too close to the diagonal.
Provide more direct evidence for the helix formation.
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 )
CD spectra of the peptide α-2 and α-2B
Conclusion The pH and ionic strength dependence of the helical
content of α-2. The change in θ222 from 0.4M to 1.6M NaCl is less than 3%.
The change in θ222 in PH 11 is less than 10%.
The change in θ222 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 CαThr40– Cδ Pro41 (native state) Cis CαThr40– Cα Pro41
Cis-trans proline isomerism
~The End