Beckman Institute. California Institute of Technology. Page 2. nascent polypeptide chain partially folded intermediates subunit mature protein aggregates.
Beckman Institute California Institute of Technology
nascent polypeptide chain
partially folded intermediates
aggregates Redrawn from FASEB J. 10, 58 (1997)
subunit
mature protein
The Nobel Prize in Chemistry 1972
Christian B. Anfinsen "for his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation"
Stanford Moore
William H. Stein
"for their contribution to the understanding of the connection between chemical structure and catalytic activity of the active centre of the ribonuclease molecule"
Levinthal’s paradox
Random search 100 amino acid protein
1.6E+27 years !
Levinthal’s pathway
What pathway(s) ??? Hydrophobic collapse
Unfolded Random coil-like
Stable secondary structure
Compact intermediate Molten globule
?
Folded Native protein
20
70
? vs.
45
20
70
probes that report on the averaged properties of the ensemble • Fluorescence
intensity • Circular Dichroism • X-ray scattering • Absorbance
Example: X-ray scattering experiment. Refolding of horse heart cytochrome c
folded
molecule
intermediate unfolded
Shuji Akiyama, Satoshi Takahashi, Tetsunari Kimura, Koichiro Ishimori, Isao Morishima, Yukihiro Nishikawa, and Tetsuro Fujisawa, Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 3, 1329-1334, February 5, 2002,
S. cerevisiae iso-1 cyt c labeled with 1,5-AEDANS at C102
DNS (C102)-cyt c
H N
CH2
CH2
NH
C
CH2
S
O
Donor (D) : HSO3
Acceptor (A) : Fe-Heme
Donor Absorbance
300
350
∞
Acceptor Absorbance
Donor Fluorescence
400
450
500
Wavelength(nm)
550
600
J ( λ ) = ∫ F D ( λ ) ε A ( λ ) λ 4 dλ 0
650
Efficiency = r06/(r06 + r6)
kET = (1/τD)(r0/r)6 r0 = 0.211(Jκ2n-4ΦD)1/6 Å 1.0
Efficiency
0.8 0.6
50 % Efficiency 0.4 0.2
r0
0.0 0
20
40
60
Distance (Å)
80
100
DNS(C102)-cyt c refolding
Unfolded
Folded
Steady state unfolding, pH 7 DNS(C102)-cytochrome c Folded ↔ Unfolded
Fraction Unfolded
1.0 0.8
fU + f F = 1
0.6 0.4
y = fU yU + fF yF
0.2
fU = (yF - y)/( yF - yU)
0.0
∆GF = ∆GF(H2O) - m[GuHCl]
∆GF (kJ/mol)
-40 -20
[GuHCl]
0
Folded: up to ~0.5 M
20
Unfolded: ~1.2 M +
40
Midpoint: ~0.85 M
60 0
1
2
[GuHCl](M)
3
Fluorescence Intensity ( 500 nm )
Refolding kinetics, pH 7, [GuHCl]=1.33 M 0.133 M DNS(C102)-cytochrome c
1
325 nm steady excitation
0.1
0.01
-2
0
2
4 6 Time(s)
8
10
I(t)
P(r)
P(k)
log(k)
I(t)
T im e
P(k)
P(r)
r (Å)
I(t)
P(k)
P(r)
T im e
log(k)
r (Å)
r (Å)
log(k)
T im e
IDA(r , t ) = I 0D exp[(−t / τD) − (t / τ )(r 0 / r ) 6 )] ∞ IDA(t ) = ∫ P(r)IDA(r , t )dr 0
6 = 1 + / 1 / τD τ D (r 0 / r ) k obs
r → ∞; k obs → 1 / τ D
Steady state unfolding, pH 7 DNS(C102)-cytochrome c 0 M GuHCl
0.23 M GuHCl
0.23 M GuHCl
0.63 M GuHCl
0.63 M GuHCl
0.84 M GuHCl
0.84 M GuHCl
1.22 M GuHCl
1.22 M GuHCl
3 M GuHCl
3 M GuHCl
P(k)
P(r)
0 M GuHCl
20 25 30 35 40 45 50 55
r(Å)
0.01
0.1
1
-1
k(ns )
10
Fluorescence Intensity
Refolding kinetics, pH 7, [GuHCl]=1.33 M 0.133 M DNS(C102)-cytochrome c
1 ms 40 ms 380 ms 760 ms 1.8 s 4.8 s 10 s 16 s
0 10 20 30 40 t Fluor
(ns)
50
t
d Fol
Refolding kinetics, pH 7, [GuHCl]=1.33 M 0.133 M DNS(C102)-cytochrome c
Hydrophobic
∆ + 1ms
∆ + 1 ms
∆ + 10 ms
∆ + 10 ms
∆ + 40 ms
∆ + 40 ms
∆ + 380 ms
∆ + 380 ms
∆ + 760 ms
∆ + 760 ms
∆ + 16 s
∆ + 16 s
Compact intermediate
Unfolded
Unfolded 60%
Compact 40%
P(k)
+
P(r)
collapse
20 25 30 35 40 45 50 55
r(Å)
0.01
0.1
1 -1
k(ns )
10
Refolding kinetics, pH 7, 0.15 M Imidazole, 1ºC, [GuHCl]=1.8 M 0.18 M DNS(C102)-cytochrome c ∆ + 1ms
∆ + 10 ms
∆ + 10ms
∆ + 60 ms
∆ + 60 ms
∆ + 180 ms
∆ + 180 ms
∆ + 760 ms
∆ + 760 ms
∆ + 5.76 s
∆ + 5.76 s
P(r)
P(k)
∆ + 1ms
20
30
40
r(Å)
50
60
0.01
0.1
1
-1
k(ns )
10
Refolding kinetics, pH 7 DNS(C102)-Co(III)-cytochrome c unfolded
2 min
2 min
3 hr 40 min
3 hr 40 min
12 hr
12 hr
17 hr 45 min
17 hr 45 min
folded
folded
P(r)
P(k)
unfolded
Tezcan et al., Proc. Natl. Acad. Sci. USA, 99, 8626-8630
20 25 30 35 40 45 50 55
r(Å)
0.01
0.1
1 -1
k(ns )
10
Idealized folding landscape (cross-section) for DNS(C102)-cytochrome c E = extended C,C’ = compact N = native
Conclusions
• Extended polypeptide conformations play an important role in DNS(C102)-cytochrome c refolding.
• These conformations may be an important means of avoiding the partially folded intermediates that can aggregate into misfolded structures, which characterize a variety of disease states.
1973 Ptitsyn: Molten globule is a kinetic intermediate on protein folding pathway
Hydrophobic collapse
Unfolded
Intermediate
Folded
Random coil-like
Molten globule
Native protein
• Compact • Native-like secondary structure • Lack of rigid tertiary structure
Molten globule formation DNS(C102)-cytochrome c
Na2SO4/H2SO4 pH 2
H2SO4 pH 2 Na2SO4
Unfolded
Intermediate
Random coil-like
Molten globule
Steady state molten globule formation, pH 2 DNS(C102)-cytochrome c 50 mM Na2SO4
100 mM Na2SO4
100 mM Na2SO4
250 mM Na2SO4
250 mM Na2SO4
330 mM Na2SO4
330 mM Na2SO4
450 mM Na2SO4
450 mM Na2SO4
700 mM Na2SO4
700 mM Na2SO4
P(r)
P(k)
50 mM Na2SO4
20253035404550556065
r(Å)
0.01
0.1
1
-1
k(ns )
10
DNS(C102)-cytochrome c Molten globule, pH 2
Refolding kinetics, pH 7
50 mM Na2SO4
∆ + 1ms
100 mM Na2SO4
∆ + 10 ms
∆ + 40 ms
P(r)
P(r)
250 mM Na2SO4
330 mM Na2SO4
450 mM Na2SO4
700 mM Na2SO4
20253035404550556065
r(Å)
∆ + 380 ms
∆ + 760 ms
∆ + 16 s
20 25 30 35 40 45 50 55
r(Å)
Conclusions
• FET kinetics provide definitive evidence for the formation of a uniformly compact molten globule at salt concentrations greater than 700 mM.
• The molten globule generated is far more homogeneous than the ensemble of polypeptides present during early cyt c refolding.
• Preliminary mapping of the protein folding landscape has been performed without any a priori assumption
• Fluorescence energy transfer kinetics technique has been applied in new ways to detect the structure and dynamics of protein folding intermediates
• Results and technique may be helpful in learning causes of various diseases
