Seminar jun 7 12

Sequence-specific control ofETS transcription factors: cause and effects

June 7, 2012Department of ChemistryGeorgia State University

Gregory PoonDepartment of Pharmaceutical Sciences

College of PharmacyWashington State University

From: HHMI, Duke University

ETS family of transcription factors

• Originally identified in transforming gene of an avian erythroblastosis virus, E26 ets = “e26 transformation specific”

• v-ets led to discovery of cellular homolog c-ets-1

• 28 known human ETS-related genes to-date

• Defined by a structurally conserved DNA-binding domain = ETS domain

Hollenhorst et al. (2011) Annu Rev Biochem 80, 437-471

Degnan et al. (1993) Nucleic Acids Res 21, 3479-3484

ETS proteins are widely distributed among animals (metazoa)

• Not found in plants, fungi, protoza

ETS is a winged helix-turn-helix domain

H1 H2

turn or “loop”

“wing”

H3

turn

H2

“wing”

Wei et al. (2010) EMBO J 29, 2147-2160.

Sequence selectivity among ETS proteins

Class III

Class I

Class II

Class IV

mEts-1 (1K79)

mElf-3 (3JTG)

mPU.1 (1PUE)

hPDEF (1YO5)

hFLI-1 (1FLI) hELK-1 (1DUX)

hSAP1-1 (1BC7)

mGABPα/β (1AWC)

PU.1, a model ETS protein• PU.1 is cellular homolog of viral oncogene Spi-1

Restricted to cells of hematopoietic and derived lineages Blood cell development, immune function, leukemia Regulates (mostly activates) expression of >50 genes

• Contains a minimal ETS domain (class III)• What is the physical basis of sequence selection?

Wei et al. (2010) EMBO J 29, 2147-160Szymczyna and Arrowsmith (2000) 37, 28363-379Poon, unpublished results

In vitro

in vivo: promoters

in vivo: ChIP-seq

Poon and Macgregor (2003) J Mol Biol, 328, 805-19Poon (2012) J Biol Chem 287, 18297-307

PU.1 ETS is a broadly sequence-selective protein

XXXGGAAYYY

AGCGGAAGTG

AAAGGAAGTG

AAAGGAATGG

AGCGAGAGTG

KD, nM

0.53 ± 0.13

2.68 ± 0.41

240 ± 70

1000

G°, kJ/mol

-4.03 ± 0.72

—

11.14 ± 0.81

14

High affinity

High affinity

Low affinity

Nonspecific

Selection of ETS binding sites

Y YYGXX GA X AAAA

CT X' C TTG G

TT

C C

Y'X'X' Y' Y'

GA

T C

AA

TT

A AA

TTTA

T

H(duplex), kJ/mol

338

312

314

299

Variants of B motif of Ig2-4 enhancer

PU.1 ETS-DNA interactions I:Structure

Sequence-specific ETS-DNA interactions

Indirect readoutProtein-phosphate contactsProtein-sugar contacts

Indirect readoutProtein-phosphate contacts

H3

turn

H2

“wing”

Direct readoutProtein-base contactsProtein-water-base contacts

Kodandapani et al. (1996) Nature 380, 456-460.

Arg235

Asn236

Arg232

T23

G9

G8

C26

C25

T24

Glu228

Lys229

5’-AAAAGGAAGTGGG-3’3’-TTTTCCTTCACCC-5’

H3

S205

K208

L250

Y252

K245

R235

5’-AAGGGGAAGTGGG-3’3’-TTCCCCTTCACCC-5’

“wing”

A

A

A

K223

N221

K219

W215

I172

R173

L174

5’-AAAAGGAAGTGGG-3’3’-TTTTCCTTCACCC-5’

H3

H2

turn or “loop”

T

T

C

A

Characterization of the protein-DNA interface in solution: footprinting

Single-hit conditions

* *

Cleavage efficiency depends on:Chemistry

AccessibilitySize of reagent

Solvent exposure of target

0.01 0.1 1 10

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

single cut

Poi

sson

pro

babi

lity

Average cut per strand

uncut

multiple cuts

KMnO4

OsO4

·OH (sugar) ·OH (sugar) ·OH (sugar)

DEPCDMS/piperidine

Biochemical probes of nucleic acids

Phosphates: DNase I, UO22+

DMS/NaOH

5’-C

TTG

GTT

TCA

CTT

CC

GC

TTAT

TT-3

’

3’-TTTATTTTCC

TTAC

CTTTG

GTTC

-5’

3’

5’

C+T DNase I protection: TTCC strand

-15 -11 -10 -9 -8 -7 -6 -50

500

1000

1500

2000

2500

3000

Inte

nsity

, AU

log [ETS, M]

-11.0-10.9 -10 -9 -8 -7 -6 -5T

T

T

A

T

T

T

T

C

C

T

T

A

C

C

T

log [ETS, M]

0.000

220.0

440.0

660.0

880.0

1100

1320

1540

1760

1980

2200

-15 -11 -10 -9 -8 -7 -6 -50

500

1000

1500

2000

Inte

nsity

, AU

log [ETS, M]

-15 -11 -10 -9 -8 -7 -6 -5

500

1000

1500

2000

2500

Inte

nsity

, AU

log [ETS, M]

High-affinity sequence

Low-affinity sequence

-10.9 -10 -9 -8 -7 -6 -5T

T

T

A

T

T

C

G

C

C

T

T

C

A

C

T

T

T

T

A

T

T

C

G

C

C

T

T

C

A

C

T

log [ETS, M]

0.000

290.0

580.0

870.0

1160

1450

1740

2030

2320

2610

2900

-15 -11 -10 -9 -8 -7 -6 -5

500

1000

1500

2000

2500

Inte

nsity

, AU

log [ETS, M]

-log [ETS, M]

Poon (2012) J Biol Chem 51, 18297-307

5’-AAAAGGAAGTGGG-3’3’-TTTTCCTTCACCC-5*’

5’-AAGGGGAAGTGGG-3’3’-TTTTCCTTCACCC-5’

Fiber model: Chandrasekaran & Arnott. (1989) The Landolt-Börnstein Database, DOI: 10.1007/10384901_24

ETS-induced DNA bending

5’-AAGGGGAAGTGGG-3’3’-TTCCCCTTCACCC-5’

-10

0

10

20

30

40

AA/TTAA/TT

AA/TT

AG/CT

GG/CC

GG/CC

GG/CC

GA/TCAA/TT

AG/CT

GT/AC

TG/C

A

GG/CC

GG/CC

4

6

8

10

12

14

16

tw

ist,

°

V

ALL,

VTI

L,

V

RO

L, °

cannonical

wid

th, Å

:

min

or g

roov

e,

maj

or g

roov

e

Poon (2012) Biochemistry 51, 4096-107

0.0 0.2 0.4 0.6 0.8 1.0

0.70

0.71

0.72

0.73

0.74

0.75

0.76

0.77

Rf(b

ound

) : R

f(free

)Flexure displacement

Poon, unpublished results

free

bound

Θ

AGCGGAAGTG Θ = 36°

Circular permutation analysis of DNA curvature

C+T DNase I protection: nonspecific sequence

GGAA strand TTCC strand

3’-TTTATTTTCTC

TAC

CTTTG

GTTC

-5’

-log [ETS, M]5

10 105

5’-*

AA

ATA

AA

AG

AG

ATG

GA

AA

CC

AA

G-3

’

0 2 4 6 8 10

0

10

20

30

40

50

Background

Bou

nd [3

2P-la

bele

d N

S o

ligo]

, pM

[NS oligo], µM

+ETS (50 nM)

KD = 1.7± 0.2 µM

Poon (2012) J Biol Chem 287, 18297-307

3’-TTTATTTTCC

TTAC

CTTTG

GTTC

-5’

DNase Ifootprinting

Gel mobilityshift

[salmon sperm DNA](mM bp)

C+T1 0.60

0.8 0.4 0.2

unbound

1:1 bound

Poon, unpublished results

0.0 0.2 0.4 0.6 0.8 1.0

0

1

2

3

4

5

6

7

Nor

mal

ized

hyp

erse

nsiti

vity

[Salmon sperm DNA], mM bp

·OH footprinting5’

-CTT

GG

TTTC

AC

TTC

CG

CTT

ATTT

-3’

-10.0 -9.3 -10.0 -9.3 -8.7 -8.0 -7.3 -6.7GCTCTAGATTTATTCGCCTTCACTTTGGTT3'

log [ETS, M]

0.3000

0.4450

0.5900

0.7350

0.8800

1.025

1.170

1.315

1.460

1.605

1.750

5'

1E-15 1E-10 1E-9 1E-8 1E-7 1E-6 1E-5

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Nor

mal

ized

inte

nsity

, AU

[ETS], M



5’-C

TTG

GTT

TCC

ATT

CC

TTTT

ATTT

-3’


-10.0 -9.3 -8.7 -8.0 -7.3 -6.7 -6.0 -5.3GCTCTAGATTTATTTTCCTTACCTTTGGTTC

log [ETS, M]

0.2000

0.3700

0.5400

0.7100

0.8800

1.050

1.220

1.390

1.560

1.730

1.900

1E-15 1E-10 1E-9 1E-8 1E-7 1E-6 1E-50.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Nor

mal

ized

inte

nsity

, AU

[ETS], M


Sequence-specific binding induces local conformational changes in DNA

G T+8

T+7

T+6

C A C T+2 T+1 C C G C T-4

T-5

A T-7

T-8

T-9

0.00.20.40.60.81.01.21.41.61.8

-3'

-3'

5'-

Cle

avag

e re

lativ

e to

unb

ound

sta

te

AGCGGAAGTG

G T+8

T+7

T+6

C C A T+2 T+1 C C T-2

T-3

T-4

T-5

A T-7

T-8

T-9

0.00.20.40.60.81.01.21.41.61.8

AAAGGAATGG

5'- H3

turn

H2

“wing”


Reactivity to MnO4-

Possible explanationsIncreased stacking?

Major groove compression?

Helical twist controls base stacking and groove widths

Randall, Zechiedrich, and Pettitt. (2009) Nucleic Acids Res 37, 5568-77

OverwoundUnderwound

Sequence-dependent control of PU.1-DNA structures

• Sequence-specific PU.1-DNA complexes are grossly similar in structure (regardless of affinity)

• Nonspecific binding is a structurally distinguishable mode of low-affinity binding

• Hypersensitivity to DNase I is a hallmark of sequence-specific ETS-DNA binding ► deformability, rather than intrinsic curvature, is important for specific sequence recognition

• Flanking sequences appear to be overwound in low-affinity specific binding ► increased DNA bending?

• Specific ETS sites load additional ETS proteins at high concentrations in a negatively cooperative manner

PU.1 ETS-DNA interactions:Thermodynamics

“Thermodynamics is a wonderful structure with no content.”

Aharon Katchalsky

Sequence-specific thermodynamics

270 280 290 300 310 320 330

14

16

18

20

22

ln (K

B, M

-1)

Temperature, K

AGC/GTG

AAA/TGG

p SHBln ln 1

C TTKR T T

AGC/GTG

AGA/GTG

AAA/GTG

ACA/GTG

AAC/GTG

TGG/TGG

-60

-40

-20

0

20

40

60

kJ/m

ol a

t 298

K

H° TS°

p H

pS

( )

ln

H C T T

TS CT

Poon and Macgregor (2004) J Mol Biol, 335, 113-27Poon (2012) Biochemistry 51, 4096-107

-2.4 -2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8

10

12

14

16

18

20

22

0.091 0.11 0.14 0.17 0.20 0.25 0.30 0.37 0.45

[Na+], M

ln (K

B, M

-1)

ln ([Na+], M)-9 -8 -7 -6 -5 -4 -3 -2 -1

AAA/GTG

AGG/GTG

ACC/GTG

AGA/GTG

AGC/GTG

CAA/GTG

TAA/GTG

GAA/GTG

AGT/GTG

AAC/GTG

AAA/TGG

NS

SKobs

Bobs +

ln ψln[M ]KSK Z

Sequence-specific thermodynamics: electrostatics

AGC/GTG

AAA/TGG

NS

Poon and Macgregor (2004) J Mol Biol, 335, 113-27Poon (2012) J Biol Chem 287, 18297-307

cocrystalstructure

-153.5

-153.0

-152.5

-152.0

-151.5

-151.0

-150.5

-150.0

-149.5

-149.0

-148.5

Pow

er,

W

0 10 20 30 40 50

-50

-40

-30

-20

-10

0

10

20

0.0 0.4 0.8 1.2 1.6 2.0ETS:DNA

1/V

dq/

d[X

]t, k

J/m

ol

[ETS], M

0 10 20 30 40 50

0.0 0.2 0.5 0.7 0.9 1.2

[DNA], M

Calorimetric measurements revealcomplex ETS-DNA interactions

Empirical fit to asymmetric dimer

k1 = 1.2 ± 0.8 nMk2 = 88 ± 34 nMh1 = -0.25 ± 0.06 kJ/molh2 = -25.2 ± 0.8 kJ/mol

H1 = -22.8 ± 0.5 kJ/molH2 ~ +16 kJ/mol

5’-AGCGGAAGTG-3’

Poon (2012) Biochemistry 51, 4096-107Poon (2010) Anal Biochem, 400, 229-36

“Forward” titrationProtein DNA

“Reverse” titrationDNA protein

-155.5

-155.0

-154.5

-154.0

-153.5

-153.0

-152.5

-152.0

-151.5

Pow

er,

W

0 10 20 30 40 50-40

-30

-20

-10

0

10

20

0.0 0.8 1.7 2.5 3.3 4.1

1/V

dq/

d[X

]t, k

J/m

ol

[ETS], M

0 10 20 30 40 50

0.0 0.2 0.4 0.7 0.9 1.1ETS:DNA

[DNA], M


k1 = 1.8 ± 1.7 nMk2 = 54 ± 38 nMh1 = +5.1 ± 0.7 kJ/molh2 = -15.2 ± 0.9 kJ/mol

H1 = -13.6 ± 0.8 kJ/molH2 ~ +12 kJ/mol

High-affinity sequence(B motif)

5’-AAAGGAAGTG-3’




-154.5

-154.0

-153.5

-153.0

-152.5

-152.0

Pow

er,

W

0 10 20 30 40 50

-10

0

10

20

300.0 0.8 1.7 2.5 3.3 4.1ETS:DNA

1/V

dq/

d[X

]t, k

J/m

ol

[ETS], M

0 10 20 30 40 50

0.0 0.3 0.5 0.8 1.1 1.3

[DNA], M

Low-affinity sequence5’-AGCGGAATGG-3’


k1 = 10-7 Mk2 = 10-6 Mh1 = +12.0 ± 0.8 kJ/molh2 = -6.7 ± 1.0 kJ/mol

H1 = +4.2 ± 0.4 kJ/molH2 ~ +12 kJ/mol

1:2




ΔG°

1:1 complex

Absence of DNA DNA present

ETS

ETS ETS

.ETSETS 2:1 complex

ETS


Sequential binding of PU.1 ETS is thermodynamically distinct

5 10 15 20 25 30 35

-40

-20

0

20

40

60

HF

, kJ

/mol

Temperature, °C

5 10 15 20 25 30 35

0

10

20

30

40

150 mM Na+

250 mM Na+

HF

, kJ

/mol

Temperature, °C


First equivalent Second equivalent

Sequence-specific thermodynamics of PU.1 ETS-DNA interactions

• Thermodynamic profile of high-affinity PU.1-DNA binding Enthalpically-driven (ΔH° < 0) Entropic penalty (ΔS° < 0) Weak salt dependence (SKobs < N) (pH-insensitive between 6.5 to 9) Osmotically destabilized

• All sequence-specific PU.1-DNA binding appears to exhibit negative ΔCp

• DNA binding modulates dimerization of PU.1 ETS

Hydration controls the sequence specificity of PU.1

Water activity, osmolality, and osmotic stress

• Interactions that involve a change in hydration are sensitive to changes in water activity “Free” (= available) water

• Experimentally accessiblethrough osmolytes

• Also biologically relevant because the cell interior is highly crowded (up to 40 g/L) Substantial fraction of water is bound to macromolecules and

metabolites (= unavailable)

wOsm 55.5 lnm a

Water activity: a tasty approach to hydration

Source: AquaLab Decagon

+ Kobs

Preferential binding vs. preferential hydration

2

2 2

obs SH O S

H O H O

ln lnln lnd K d ad a d a

2H O 2 SA + B AB + H O + S Kobs

Kobs

Sensitivity to osmotic stress

2

2 2

obs SH O S

H O H O

ln lnln lnd K d ad a d a

2H O 2 SA + B AB + H O + S Kobs

N+O

O-HOOHO

OHO

HO

OH

HO

HO

OH

OH

OH

OO

OH

H

H

H

HH

HH

NH

N OH

I II III IV

Poon (2012) J Biol Chem 287, 18297-307

Poon (2012) J Biol Chem 287, 18297-307

●: ̶ osmolyte○: + osmolyte

PU.1-DNA complexes respond differentially to osmolytes

0 1 2 3 4 5 6

12

14

16

18

20

22

1.0 0.98 0.96 0.95 0.93 0.91 0.90

Water activity (aw)

ln (K

B, M

-1)

Osmolality

AGCGGAAGTG

AAAGGAATGG

AGCGAGAGTG

Poon (2012) J Biol Chem 287, 18297-307

Perturbation of PU.1-DNA complexes by osmolytes

○ TEG Betaine Sucrose▲ Nicotinamide

PWB B

w

ln ln Osm 55.5 ln 55.5K K

a

Poon (2012) J Biol Chem 287, 18297-307

G+A

5’-A

AAT

AA

GC

GG

AA

GTG

AA

AC

CA

AG

-3’

3’-GA

AC

CA

AA

GG

TAA

GG

AA

AATA

AA

-5’

High-affinitysequence

Low-affinitysequence

Poon (2012) J Biol Chem 287, 18297-307

DMS protection: GGAA strand

Poon (2012) J Biol Chem 287, 18297-307

337 Å3 / ~30 Å3 per H2O = ~11 H2O

Hydration in PU.1 ETS-DNA interactions

• Osmolytes discern 3 binding modes for PU.1-DNA binding: Net uptake ► high-affinity Net neutral ► low-affinity specific Net release ► nonspecific

• Lack of dependence on osmolyte identity for sequence-specific complexes implies a steric effect on water Core consensus is a water-sequestering cavity Accessible to bulk solvent in the low-affinity inaccessible in the high-affinity ETS-

DNA complex

• OS experiments predict a larger water uptake than structurally available Linked uptake of osmolyte? (Unlikely) ΔΓPW is an effective quantity, not necessarily a stoichiometry Immobilized waters form a highly cooperative hydration network

W

.ETSETS

W

Sequence-specific ETS recognition: a model

W WW W W

High-affinity binding Low-affinity binding

Extensive interfacial hydrationStrongly destabilized to osmotic stress

Water-sequestering core cavityProtection from DNase I, DMS, and ·OH

Enthalpically driven bindingImmobilize water network at entropic cost

Weak salt dependence

Small induced DNA curvatureLess overwound flanking segments

Poorly hydrated interfaceInsensitive to osmotic stress

“Leaky” core cavityProtection from DNase I and ·OH but not DMS

Entropically driven bindingIncreased backbone contacts (electrostatic)

Strong salt dependence

Pronounced induced DNA curvatureOverwound flanking segments

AGCGGAAGTG AAAGGAATGG

.ETSETS

W

Acknowledgements

Victor M. Bii

CollaboratorsDr. W. David Wilson (Chemistry,

Georgia State University)Dr. Arjan van der Vaart (Chemistry,

University of South Florida)

Financial supportAmerican Cancer Society (IRG -77-003-27)College of Pharmacy, WSU

Extra slides

Sequence-specific protein monomers, although not infrequent, have little to

contribute to DNA bending.

Dickerson and Chiu (1998) Biopolymers 44, 361-403

Quantitative footprinting: methodology

ETS

-bou

nd

A

Unb

ound

5'-AAATAAGCGGAAGTGAAACCAAG-3'

5'-AAATAAGCGGAAGTGAAACCAAG-3'5'-CTTGGTTTCACTTCCGCTTATTT-3'

5'-CTTGGTTTCACTTCCGCTTATTT-3'

0( )i i iQ a P x dx

Reactivity Q at position i is proportional to integrated intensity of corresponding peak centered at x = xi

2 20

1 γπ ( ) γiQ x x

1

n

ii

Q Q

Using a Lorentzian distribution to describe the line shape of Pi,

The trace is taken as a linear combination of individual band reactivities:

0 1000 2000 3000 4000 5000 6000

-155

-154

-153

-152

-151

-150

-149

-148

0 1000 2000 3000 4000 5000 6000

-155

-154

-153

-152

-151

-150

-149

-148

0 1000 2000 3000 4000 5000 6000

-155

-154

-153

-152

-151

-150

-149

-148

0 1000 2000 3000 4000 5000 6000

-155

-154

-153

-152

-151

-150

-149

-148

0 1000 2000 3000 4000 5000 6000

-155

-154

-153

-152

-151

-150

-149

-148

0 1000 2000 3000 4000 5000 6000

-155

-154

-153

-152

-151

-150

-149

-148

0 1000 2000 3000 4000 5000 6000

-155

-154

-153

-152

-151

-150

-149

-148

Nor

mal

titra

tion

(ETS

into

DN

A)

1:11:1

1:1

1:21:1

2:1

2:1

1:2 1:2

Rev

erse

titra

tion

(DN

A in

to E

TS)

ETS into buffer

AAAGGAATGGAAAGGAAGTG

Pow

er,

W

25°C, 150 mM Na+

time, s

AGCGGAAGTG

exothermic


ETS family of transcription factors

• Hematopoiesis• Organ cell fate• Neuronal development

Hollenhorst et al. (2011) Annu Rev Biochem 80, 437-471

YXETS

Sequence-specific control ofETS interactions: a model

NucleusExcess DNA, molecular confinement, crowding

ETS

ETSX

Cytosol

Binding site A Binding site B

X

H3

5’

5’

GGAA strand

TTCC strand

Dnase I hypersensitivity is stereospecific

5’-*

AA

ATA

AG

CG

GA

AG

TGA

AA

CC

AA

G-3

’ 3’-GA

AC

CA

AA

GG

TAA

GG

AA

AATA

AA

*-5’

C+T

3’

5’

-15 -11 -10 -9 -8 -7 -6 -5

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

, AU

log [ETS, M]

-11 -10 -9 -8 -7 -6 -5A

T

A

A

G

C

G

G

A

A

G

T

G

A

A

A

A

T

A

A

G

C

G

G

A

A

G

T

G

A

A

A

log [ETS, M]

200.0

555.0

910.0

1265

1620

1975

2330

2685

3040

3395

3750

-11.0-10.9 -10 -9 -8 -7 -6 -5A

T

A

A

A

A

G

G

A

A

T

G

G

A

A

A

A

T

A

A

A

A

G

G

A

A

T

G

G

A

A

A

log [ETS, M]

200.0

450.0

700.0

950.0

1200

1450

1700

1950

2200

2450

2700

-15 -11 -10 -9 -8 -7 -6 -5

500

1000

1500

2000

2500

3000

Inte

nsity

, AU

log [ETS, M]



DNase I protection: GGAA strand-log [ETS, M]55

10 10

Reciprocal ETS-DNA equilibria: a scheme



DNA binding is negatively cooperative withrespect to protein dimerization

ETS dimerization is negatively cooperative withrespect to DNA binding

P: PU.1 ETSD: DNA


Unbound PU.1 ETS is a weak dimer

0 2 4 6 8 10 12 14 16 18

0.026

0.028

0.030

0.032

0.034

0.036

(K C

)/R,

kDa-1

C, g mL-1

Apparent MW: 27.5 ± 1.1 kDaFormula weight: 13.0 kDaApparent stoichiometry: 2.1 ± 0.1(Concentration: 0.9 mM)

SLS DLS

24.4 kDa


Unbound PU.1 ETS is a weak dimer

0 5 10 15 20 25

18.2

17.7

18 µM

Elution volume, mL

130 µM

-6.0 -5.5 -5.0 -4.5 -4.0 -3.5

17.4

17.6

17.8

18.0

18.2

Elu

tion

volu

me,

mL

log (PU.1 ETS, M)

Poon (2012) Biochemistry 51, 4096-107 and unpublished results

KD ~ 20 µM

SEC

ETS-DNA interactions: summary

• The PU.1 ETS domain assumes different oligomeric states in the unbound vs. DNA-bound states.

• These oligomeric states are thermodynamically distinguishable, implying possible distinct conformations between the free and bound states.

• Conformational changes induced by DNA binding may represent specific permissive or inhibitory states for protein-protein interactions.

-6 -4 -2 0 2 4 6-6

-4

-2

0

2

4

6

8

AGC

ACC

AGG

TAC

AGT

ACT ACT

TGA

G

° fro

m A

AA

G°int

from AAA

AAA AXA

AAY AXY

ΔΔG° (AXA)

ΔΔ

G°

(AAY

) ΔΔG° (AXY)

Cooperative coupling by flanking bases

AAAGGAAGTG

int AXY AXA AAY AAA( )G G G G G

Poon and Macgregor (2003) J Mol Biol, 328, 805-19

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