chemical exchange and ligand binding -...

Chemical Exchange

and Ligand Binding

• NMR time scale

• Fast exchange for binding constants

• Slow exchange for tight binding

• Single vs. multiple binding mode

• Calcium binding process of calcium binding

proteins

• CaM regulation of gap junctions

Effects of Chemical Exchange on NMR Spectra

•Chemical exchange refers to any process in which a nucleus

exchanges between two or more environments in which its NMR

parameters (e.g. chemical shift, scalar coupling, or relaxation) differ.

•DNMR deals with the effects in a broad sense of chemical exchange

processes on NMR spectra; and conversely with the information

about the changes in the environment of magnetic nuclei that can be

derived from observation of NMR spectra.

Conformational

equilibrium

Chemical

equilibrium

Kex

KB

Types of Chemical Exchange Intramolecular exchange

– Motions of sidechains in proteins

– Helix-coil transitions of nucleic acids

– Unfolding of proteins

– Conformational equilibria

Intermolecular exchange

– Binding of small molecules to macromolecules

– Protonation/deprotonation equilibria

– Isotope exchange processes

– Enzyme catalyzed reactions

A B

M+L ML

Because NMR detects the molecular motion itself, rather the numbers

of molecules in different states, NMR is able to detect chemical

exchange even when the system is in equilibrium

Typical Motion Time Scale for

Physical Processes

NMR Time Scale

Time Scale Chem. Shift, d Coupling Const., J T2 relaxation

Slow k << dA- dB k << JA- JB k << 1/ T2,A- 1/ T2,B

Intermediate k = dA - dB k = JA- JB k = 1/ T2,A- 1/ T2,B

Fast k >> dA - dB k >> JA- JB k >> 1/ T2,A- 1/ T2,B

Sec-1 0 – 1000 0 –12 1 - 20

• NMR time-scale refers to the chemical shift timescale.

• The range of the rate can be studied 0.05-5000 s-1 for H

can be extended to faster rate using 19F, 13C and etc.

2-state First Order Exchange

Lifetime of state A:

tA = 1/k+1

Lifetime of state B:

tB = 1/k-1

Use a single lifetime

1/ t =1/ tA + 1/tB

= k+1+ k-1

A B k1

k-1

Rationale for Chemical Exchange

For slow exchange

For fast exchange

Bloch equation approach:

dMAX/dt = -(DωA)MAY - MAX/tA + MBX/tB

dMBX/dt = -(DωB)MBY - MBX/tB + MAX/tA

•

•

•

FT

FT

2-state 2nd Order Exchange

Kd = [M] [L]/[ML] = k-1/k+1

M+L ML k+1

k-1

Kd =10-3 – 10-9 M

kon = k+1 ~ 108 M-1 s-1 (diffusion-limited)

k-1 ~ 10-1 – 10-5 s-1

Lifetime 1/ t =1/ tML + 1/tl

= k-1 (1+fML/fL) fML and fL are the mole fractions of bound and free ligand,

respectively

Slow Exchange k << δA -δB

• Separate lines are observed for each state.

• The exchange rate can be readily measured

from the line widths of the resonances

• Like the apparent spin-spin relaxation rates,

1/T2i,obs

1/T2A,obs= 1/T2A+ 1/tA = 1/T2A+ 1/k1

1/T2B,obs= 1/T2B+ 1/tB = 1/T2B+ 1/k-1

line width Lw = 1/pT2 = 1/pT2 + k1/p

Each resonance is broadened by D Lw = k/p

Increasing temperature increases k, line width

increases

A B k1

k-1

Slow Exchange for M+L ML

• Separate resonances potentially are observable for both the free and bound states MF, MB, LF, and LB

• The addition of a ligand to a solution of a protein can be used to determine the stoichiometry of the complex.

• Once a stoichiometric mole ratio is achieved, peaks from free ligand appear with increasing intensity as the excess of free ligand increases.

• Obtain spectra over a range of [L]/[M] ratios from 1 to 10

k1

k-1

Slow Exchange for M+L ML

• For free form 1/T2L,obs= 1/T2L+ 1/tL = 1/T2L+ k-1 fML/fL

1/T2M,obs= 1/T2M+ 1/tM = 1/T2M+ k-1 fML/fM

For complex form

1/T2ML,obs= 1/T2ML+ 1/tML = 1/T2ML+ k-1

Measurements of line width during a titration can be used to derive k-1 (koff).

k1

k-1

19F spectra of the enzyme-inhibitor complex at

various mole ratio of carbonic anhydrase:inhibitor

• At -6 ppm the broadened peak for the bound ligand is in slow exchange with the peak from free ligand at 0 ppm.

• The stoichiometry of the complex is 2:1. No signal from the free ligand is visible until more than 2 moles of inhibitor are present.

1:0.5

1:4

1:3

1:2

1:1

Free inhibitor Bound ligand

Coalescence Rate

• For AB equal concentrations, there will be a rate of interchange where the separate lines for two species are no longer discernible

• The coalescence rate

Dd is the chemical shift difference between the two signals in the unit of Hz.

Dd depends on the magnetic field

kc = p Dd / √2 = 2.22 Dd

Coalescence Temperature

• Since the rate depends on

the DG of the inversion,

and the DG is affected by

T, higher temperature will

make things go faster.

• Tc is the temperature at

which fast and slow

exchange meet.

• T>Tc, fast exchange

• T<Tc, slow exchange

T TC

we can calculate the DG‡ of the process

DG‡ = R * TC* [ 22.96 + ln ( TC / Dd ) ]

Fast Exchange k >> δA -δB

• A single resonance is observed, whose

chemical shift is the weight average of the

chemical shifts of the two individual states

δobs = fAδA +fBδB, fA + fB = 1

For very fast limit

1/T2,obs= fA/T2A+ fB/T2B

For moderately fast

1/T2,obs= fA/T2A+ fB/T2B + fAfB2

4p (DdAB)2/ k-1

Maximal line broadening is observed when

fA = fB = 0.5

A B k1

k-1

Fast Exchange k >> δA -δB

For M δM,obs = fMδM +fMLδML

For L δL.obs = fLδL +fMLδML

1/T2,obs= fML/T2,ML+ fL/T2,L + fMLfL2

4p

(dML-dL)2/ k-1

• A maximum in the line broadening of ligand or protein resonances occurs during the titration at a mole ratio of approx. ligand:protein 1:3

• The dissociation constant for the complex can be obtained by measuring the chemical shift of the ligand resonance at a series of [L].

M+L ML k+1

k-1

Identification of Ca2+

binding sites in ECD of

CaSR

How can CaSR sense

the change of Ca2+o

within a narrow range?

(multiple sites?

cooperativity?)

Identification of

CaM binding region

in c-tail of CaSR

Integration of Calcium Signaling Via CaSR

Y. Huang, JJ Yang, J Biol Chem. 2007; Yun Huang.. JJ Yang Biochemistry 2009; Y Hang, … JJ Yang, JBC 2010

LB1

LB2Site 1Site 1

Site 4Site 4

Site 3Site 3Site 2Site 2

site5site5

site1

site3 site2

LB

1

LB2

site3

site2

site4

site5

Yun Huang.. JJ Yang Biochemistry 2009

Subdomain Approach

0

0.2

0.4

0.6

0.8

1

0 1 2 3 4 5 6 7Re

lative

cha

ng

e o

f che

mic

al shift

[Ca2+

] (mM)

Two Distinct Ca2+-Binding Processes Revealed by NMR

site1site1

site3site3site2site2

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35

Rela

tive

cha

ng

e

[Ca2+

] mM

0.0

2.0 105

4.0 105

6.0 105

8.0 105

1.0 106

1.2 106

440 460 480 500 520 540 560 580 600

Flu

ore

sce

nce

Inte

nsity

wavelength (nm)

Kd1 = 0.7 ± 0.1 mM

Kd2 = 6.4 ± 0.8 mM

n hill = 2.9 ± 1.2

Kd: 1.6 ± 0.1 mM

nHill: 2.3 ± 0.3

ANS + Protein + Ca2+

ANS + Protein

ANS

Yun Huang.. JJ Yang Biochemistry 2009;

Developing Calcium Sensors by Design

1. Highly targeting specificity

2. Simple stoichiometric

interaction mode to ease

calibration 3. Tunable affinities, selectivity

& kinetics

4. Minimal perturbation on

signaling without using

natural calcium binding

proteins

JACS, 2002, 2005, 2007 Biochem, 2005, 2006, PEDS, 2007,

protein science 2008

J. Zhou, A. Hofer, J.J. Yang, Biochem, 2007

A.Holder, … J.J. Yang, Biotech 2009

S. Tang,….O. Delbano J.J. Yang, PNAS, 2011

0

0.3

0.6

0.9

1.2

1.5

250 300 350 400 450 500 550

EGFPD8D9D10CatchERD12

No

rmalized

ab

so

rban

ce

Wavelength (nm)

0

0.2

0.4

0.6

0.8

1.0F488; 10 M EGTA

F488; 5 mM Ca2+

F395; 10 M EGTA

F395; 5 mM Ca2+

No

rmalized

flu

ore

scen

ce

Ca2+

x- x-

x- x-

x-

Ca2+

R

O-

R

OH

Y66 Y66

Neutral Anionic

Catcher: Ca2+ Sensor for Detecting High Concentration

chromophore

Designed Ca2+ binding site 0

0.2

0.4

0.6

0.8

1.0

500 520 540 560 580 600

No

rmalized

flu

ore

scen

ce

Wavelength (nm)

0

0.1

0.5

1.0 6.0 mM Ca2+

100

80

60

40

20

0

No

rma

lize

d f

luo

resc

en

ce (

%)

E225

D202

E223

E204

E147


CatchER: Ca2+ binding capability

1.5

2.0

2.5

3.0

3.5

10 15 20 25 30

F488

F395

A488

Rela

tive a

mo

un

t o

f C

a-b

ou

nd

Catc

hE

R

[CatchER] M

0

0.2

0.4

0.6

0.8

1.0

500 520 540 560 580 600

No

rmalized

flu

ore

scen

ce

Wavelength (nm)

Equilibrium dialysis

coupled with ICP-OES fluorescence

1H, 15N-HSQC


0

20

40

60

80

100

0 0.02 0.04 0.06 0.08 0.1

no

rmalized

flu

ore

scen

ce (

%)

time (s)

0

20

40

60

80

100

0 0.1 0.2 0.3 0.4 0.5

no

rmalized

flu

ore

scen

ce (

%)

time (s)

1000

500

300

200

100

50

0

[Ca2+] µM

0

200

[EGTA]

µM

Fast Kinetics: koff = 700 s-1

Fast Kinetics of CatchER

kon = koff/Kd= 3.9 x 106 M-1s-1


CatchER’s Ca2+ Induced Chemical Shift Changes

Y182 G228


Chemical Exchange in Rat CaM 0:

1, C

a:C

aM1:

1, C

a:C

aM2

:1, C

a:C

aMa

G113

G113

G96

G59

G23

b

Fast Exchange

Slow Exchange

Fast

Slow

Intermediate

I II

II

I

I

V

Rat CaM

Indicates:

Initial occupancy of C-

terminal domain

Suggests:

Temporal occupancy of N-

terminal domain at low [Ca]

and/or domain coupling

Kirberger M, Yang JJ, JIBC, 2013.

Similarities in Chemical Exchange with Ca2+ Titration

Jaren, O.R., et al., Calcium-induced conformational switching of Paramecium calmodulin provides evidence for domain coupling. Biochemistry, 2002. 41(48): p. 14158-66. Sun, H., et al., Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin. Biochemistry, 2001. 40(32): p. 9605-

17.

Slow exchange

Fast exchange

No exchange

Slow then fast exchange

Paramecium

CaM

I II

II

I

I

V

Rat CaM

Slow exchange

Fast exchange

intermediate exchange

Kirberger M, Yang JJ, JIBC, 2013.

The Connexin Family Tree

Söhl et al., Nat. Rev. Neurosci. 6: 191-200, 2005

33

Identifying CaM Binding Region in Gap

Junction Connexins

Cx50_m

Cx46_h

Cx44_s

Score

Cx43_h

xxBxB#xxx#xxxx#xxx

NH2

COOH

1 5 10

CaMKI_h

CaMKII_h

789999999999999876

145 TKKFRLEGTLLRTYVCHI 162

789999999999987654

136 RGRVRMAGALLRTYVFNI 153

899999999999766543

133 RGKVRIAGALLRTYVFNI 150

479999999999999999

142 HGKVKMRGGLLRTYIISI 161

000999999999999999

294 FAKSKWKQAFNATAVVRH 315

000999999999999999

289 NARRKLKGAILTTMLATR 310

Y. Zhou. JJ Yang JBC. 2007; Zhou Y, Yang JJ. Biophys J. 2009 ; Y. Chen, .. JJ Yang Biochem J. 2011

Monitoring Cx Peptide and Calmodulin

[Cx43]/[CaM]

0:1 0.4:1 0.8:1 1.2:1

-0.2

-0.1

0

0.1

0.2

0 0.5 1 1.5 2

K94D64G33G61G25K148A57

Dp

pm

-H

[Cx43]/[CaM]9.0 9.1 9.2

9.0 9.1 9.2

1H (ppm)

113

114

115

113

114

115

113

114

115

113

114

115

113

114

115

113

114

115

[Cx44]/[CaM]

0:1

0.3:1

0.6:1

0.9:1

1.2:1

2.0:1

1H (ppm)

15N

(ppm

)

132

133

134

8.3 8.4 8.5

132

133

134

132

133

134

132

133

134

132

133

134

132

133

134

8.3 8.4 8.5

T29

T117

A57

T29

T117

A57

35

Y. Zhou. JJ Yang JBC. 2007

8.70 8.65 8.60 8.55

CaM : Cx50p

1 : 0

1 : 0.4

1 : 1

1 : 1.2

8.70 8.65 8.60 8.55

8.70 8.65 8.60 8.55

8.70 8.65 8.60 8.55

105.2

105.2

105.2

105.2

Free G33

bound

105.4

105.4

105.4

105.4

105.6

105.6

105.6

105.6

Strong Binding Indication by Slow Exchange

CaM +

Cx43p

36

Y. Chen, .. JJ Yang Biochem J. 2011

G33

G134 T29

T117

I27

I100

V136

D64 A57

I130

K148

K21

K94

A147

Holo-CaM Holo-CaM + Cx50p

N137

L116

A128

F19 T70

HSQC Spectra of Holo-CaM with Cx50 Peptide

37

Y. Chen, .. JJ Yang Biochem J. 2011

chemical exchange and ligand binding -...

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