Chemical Exchange and Ligand Binding -...
Transcript of 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
S. Tang,….O. Delbano J.J. Yang, PNAS, 2011
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
S. Tang,….O. Delbano J.J. Yang, PNAS, 2011
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
S. Tang,….O. Delbano J.J. Yang, PNAS, 2011
CatchER’s Ca2+ Induced Chemical Shift Changes
Y182 G228
S. Tang,….O. Delbano J.J. Yang, PNAS, 2011
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