Download Conformational Analysis of a Set of Peptides Corresponding

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
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℃
206nm 222nm
β1:1-11 β2:12-23 α1: 24-34 β3:35-42
α2:40-56
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)
3J
HNCα:● : below 6 HZ (α-helix)
○ : above 8 HZ(β-sheet)
－ : not measure
: 6-8 HZ(random coli)
The α-2 Peptide NMR Spectrum
PH 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)
α-2B: 41-56 (32% helix )
PH 5.4 4℃
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