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