Non-cooperative thermal denaturation of acyl-CoA binding protein

Tetsu KIMURA

10th Group MeetingJuly 8, 2009

California Institute of Technology

-Purpose-Molecular Mechanism of Protein Folding

Bryngelson, Onuchic & Wolynes, Proteins (1995) 21, 167

U

I

N

The determination of the factors that control the assembly of structural components.

Quantitative estimates of the changes in energiesCharacterization of the populated structures along the folding pathway

-Introduction & Purpose-Denatured State Ensemble

Bryngelson, Onuchic & Wolynes, Proteins (1995) 21, 167

U

The determination of the structures of the denatured state ensemble is requiredas the starting point of the reaction to understand the protein folding mechanism.

Hoffmann A. et.al. PNAS;2007;104:105-110

-Previous Studies-Single-molecule FRET measurements

-Result-Static FET Kinetics

1.0

0.8

0.6

0.4

0.2

0.0Norm

aliz

ed F

luor

esce

nce

Inte

nsity

40x10 -93020100Time (sec)

10 15 20 25 30 35 40 45 50 55

10 15 20 25 30 35 40 45 50 55

r (Å)

r (Å)

34.7 Å

NativeState

UnfoldedState

(3M GuHCl)

26.8 Å

I(t) = P(k) exp(–kt)k

1k(r) = 1k0 r0

r

6

1 +

U

N

rh Donor

Acceptor

FET kinetics is the best method to monitorthe distance distribution experimentally.

-Pair of Donor and Acceptor- IAEDANS and QSY®35

1,5-IAEDANS (DNS)

NHCH2CH2NH C

O

CH2

SO3H

CH2NH C

O

CH2

NH

NO2

N

NO

QSY®35

475 = 24,000 cm-1 M-1

No mission

R0 = ~35 Å

25000

20000

15000

10000

5000

0

(M

-1·c

m-1

)

700650600550500450400Wavelength (nm)

1.0

0.8

0.6

0.4

0.2

0.0

Norm

alized Fluorescence Intensity

Cys

Cys

DNS-QSY pair is a sensitive probe for conformational changes.

(10.5 < r < 52.5 Å)

2

16 8621

3862 66

50

-Target Protein-Acyl-CoA Binding Protein (ACBP)

Four-helix bundle86 residues10 kDa

81716151413121111Number of Residues

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

Hyd

roph

obic

ity

100

80

60

40

20

0

Hel

ix P

rope

nsity

81716151413121111Number of Residues

Helix-1Helix-2Helix-3Helix-4

Helix-1 /Helix-4

Q2-K16D21-D38K50-K62K66-I86

Q2-K66K16-I86

25.725.122.825.6

11.05.7

42463949

8791

D(C-C), Å <RCDrms>, Å

Double-Cys Mutants

ACBP is a good model for both experiment and simulation.Kyte & Doolittle (1982) JMB 157, 105 Muñoz & Serrano (1995) JMB 245, 275

-Method-How to label the proteins

Stock solutionof unlabeled ACBP(0.4 – 1mM)

DTT Incubate30 min

Desalting column to remove DTT& change the buffer (3 M GuHCl, pH 8.0)

Dyes

Desalting column to refold(20 mM Tris-HCl (pH 8.0) + x mM NaCl)

monoQ column to purify(20 mM Tris-HCl (pH 8.0) with NaCl gradient)

TCEP

-Previous Study-Purification and Static Properties of Double-labeled ACBP

0.06

0.05

0.04

0.03

0.02

0.01

0.00

Abs

orba

nce

700600500400300Wavelength (nm)

2.0x106

1.5

1.0

0.5

0.0

Fluo

resc

ence

Inte

nsity

700650600550500450400Wavelength (nm)

DNS fluorescence is highly quenched by QSY®35 in the native state.

NU in 3.0 M GuHCl

UV-vis & Fluorescence spectra

Abs

orpt

ion

300250200150100500Elution (mL)

25

20

15

10

5

0

%B

Abs

orpt

ion

120100806040200Elution (mL)

25

20

15

10

5

0

monoQ purification of DNS-labeled ACBP

monoW purification of DNS & QSY-labeled ACBP

2-16

2(DNS)-16

2-16(DNS)

2(DNS)-16(DNS)

2(QSY)-16(DNS)

280 nm

355 nm

490 nm

280 nm355 nm

490 nm

2-66

-25

-20

-15

-10

-5

0

[22

2] (x

103 d

eg·c

m2 ·d

mol

-1)

8070605040302010Temperature (ºC)

-Result-Temperature Melting Followed by CD

-25

-20

-15

-10

-5

0

[22

2] (x

103 d

eg·c

m2 ·d

mol

-1)

8070605040302010Temperature (ºC)

WTWT* (W55F)662-6666-86

Double-labeled ACBPs are as stable as single-labeled ACBP.

2(QSY)-66(DNS)

66(DNS)-86(QSY)

-Result-Thermal Melting by Fluorescence Spectra

2(QSY)-66(DNS)

11.0 Å

2.5

2.0

1.5

1.0

0.5

0.0

Fluo

resc

ence

Inte

nsity

(/10

6 )

700650600550500450400Wavelength (nm)

10 ℃

80 ℃

DNS is partially buried from the solvent in the native state.

515 nm

-25

-20

-15

-10

-5

0

[22

2] (x

103 d

eg·c

m2 ·d

mol

-1)

8070605040302010Temperature (ºC)

3.0x106

2.5

2.0

1.5

1.0

0.5

0.0

Fluorescence Intensity

-Result-Thermal Melting of Labeled ACBPs

11.0 Å(~87 Å)

2(QSY)-66(DNS) 66(DNS)-86(QSY)

25.6 Å(~49 Å)

-25

-20

-15

-10

-5

0

[22

2] (x

103 d

eg·c

m2 ·d

mol

-1)

8070605040302010Temperature (ºC)

3.0x106

2.5

2.0

1.5

1.0

0.5

Fluorescence Intensity

-25

-20

-15

-10

-5

0

[22

2] (x

103 d

eg·c

m2 ·d

mol

-1)

8070605040302010Temperature (ºC)

3.0x106

2.5

2.0

1.5

1.0

0.5

0.0

Fluorescence Intensity

Non-cooperative melting of helix-4

-Result-Distance Distributions of Native and Unfolded States

A bimodal distritbuion of native and denatured states for 2-66.66-86 does not show the cooperative folding.

20 ℃

r (Å)

10 15 20 25 30 35 40 45 50 550

0.1

0.2

0.3

0.4

0.5

10 15 20 25 30 35 40 45 50 550

0.1

0.2

0.3

0.4

0.5

10 15 20 25 30 35 40 45 50 550

0.1

0.2

0.3

0.4

0.5

10 15 20 25 30 35 40 45 50 55

49 ℃

75 ℃

2(QSY)-66(DNS) 66(DNS)-86(QSY)

10 15 20 25 30 35 40 45 50 550

0.05

0.1

0.15

0.2

0.25

20 ℃

45 ℃

10 15 20 25 30 35 40 45 50 550

0.05

0.1

0.15

0.2

0.25

10 15 20 25 30 35 40 45 50 550

0.05

0.1

0.15

0.2

0.25

75 ℃

r (Å)

10 15 20 25 30 35 40 45 50 55

-Discussion-Cooperative or Non-cooperative Folding?

U

N N

U

Sadqi, Fushman, Muñoz Nature 442, 317-321 (2006)

10 15 20 25 30 35 40 45 50 550

0.1

0.2

0.3

0.4

0.5 N U

-Result-Residual Structures in the Unfolded States

Unfolded Statein 6.0 M GuHCl

Unfolded Statein 2.1 M GuHCl

Native Statein 0.25 M GuHCl

10 15 20 25 30 35 40 45 50 550

0.05

0.1

0.15

0.2

0.25

10 15 20 25 30 35 40 45 50 550

0.05

0.1

0.15

0.2

0.25

10 15 20 25 30 35 40 45 50 550

0.05

0.1

0.15

0.2

0.25 10 15 20 25 30 35 40 45 50 550

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

10 15 20 25 30 35 40 45 50 55r (Å)

r (Å)

10 15 20 25 30 35 40 45 50 55

20 ℃

45 ℃

75 ℃

66(DNS)-86(QSY)25.6 Å (~49 Å)