Download Average protein synthesis time

Bacterial ribosome
Exit tunnel: 100 Å x 20 Å
30-40 aa
N
Netzer & Hartl (1997)
Nature 388:343
Average protein synthesis
time
Co-translational Folding Model
• Prokaryotes (~6 sec)
– ~300 amino acids
– Translation rate (~50 aa/sec)
• Eukaryotes (~2 min)
– ~500 amino acids
– Translation rate (5 aa/sec)
Fedorov and Baldwin (1997) J. Biol. Chem. 272:32715
Funnel Model of Co-translational Folding
bacteria
Fedorov and Baldwin, JBC (1997)
Bukau et al., (2000) Cell 101:119
Fedorov and Baldwin (1997) J. Biol. Chem. 272:32715
Trigger Factor (TF)
•
•
•
•
E. Coli
PPI activity (FKBP)
Chaperone-like activity
X-linked to ribosome-arrested chains 57
aa
A. Horwich (2004) Nature 431:
Eukaryote
Bukau et al., (2000) Cell 101:119
Purvis et al., (1987) J. Mol. Biol. 193:413-417.
Hartl & Hayer-Hartl (2002) Science 295:1852-58.
Nascent chains resolved by 2D SDS-PAGE
Oxidation of HA
165
Time (h) 1
IT1
IT2
NT
81
2
#1
4
-DTT
IT1
Non-reducing
IT2
NT
38
285
R
22
Reducing
N
483
8
+DTT
#2
C
Chen et al., (1995) PNAS
Fluoresence resonance energy transfer (FRET)
Efficiency of transfer depends on:
• Extent of overlap of the donor emission and
the acceptor absorption spectra
• Relative orientation of the donor (D) and
acceptor (A)
• Distance of the donor and acceptor
• E = R06 / (R06 + R6)
• R0 = distance at which the energy of transfer from
D to A is 50% (typically 20-60 Å)
R is the distance separating the two dyes.
Selvin (2000) Nat. Struct. Biol. 7:730.
Incorporation of a modified Lysine into a nascent chain
Woolhead et al., (2004) Cell 116:725-736.
Woolhead et al., (2004) Cell 116:725-736.
Woolhead et al., (2004)
Cell 116:725-736.
Woolhead et al., (2004) Cell 116:725-736.
Photocrosslinking
to
ribosomal proteins
Woolhead et al., (2004) Cell 116:725-736.
Woolhead et al., (2004) Cell 116:725-736.
Proteases protect
themselves by having Prosequences
• Prevent premature activation
• Provide kinetic stability
(act as a foldase)
!-lytic protease
• Bacterial protease secreted by gramnegative bacterium Lysobacter
enzymogens to degrade other soil
microorganisms
• Family of serine proteases including
chymotrypsin, trypsin and elastase.
• 198-aa with a 166-aa N-terminal pro
region (Pro) required for folding added
cis or trans.
!-lytic Folding
• without Pro region
– initial rate 1.18 x 10-11 s-1
– t 1/2= ~1,800 years
– folding barrier 30 kcal/mol
• with Pro region
– initial rate 0.037 s-1
– t 1/2= ~30 s
– folding barrier 18 kcal/mol
Sol et al., (1998) Nature 395:817
Pro Region
Cunningham et al., (1999) PNAS 96:11008
Cunningham et al., (1999) PNAS 96:11008
Cunningham et al., (1999) PNAS 96:11008
BPTI
-10
proN’
TPGCDTSNQAKAQ
[30-51]
very
fast
fast
PRE
PRO
MATURE
30-51
14-38
med -10SSG med
PRO
proR
Pre-Pro-BPTI
fast
Pro-BPTI
30-51
14-38
proN’
[-10SSG]
PRO
MATURE
[5-55]
very
fast
5-55
14-38
proN*
Weissman & Kim (1992) Cell 71:841-851.
proN
III
30-51
5-55
med 14-38