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![Page 1: Florida State University, National High Magnetic Fields Laboratory Piotr Fajer Conformational Changes Associated with Muscle Activation and Force Generation.](https://reader035.fdocuments.us/reader035/viewer/2022062715/56649da55503460f94a907e5/html5/thumbnails/1.jpg)
Florida State University,
National High Magnetic
Fields Laboratory
Piotr Fajer
Conformational Changes Associated with Muscle Activation
and Force Generation by Pulsed EPR Methods
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Motor proteinsMotor proteinsMotor proteinsMotor proteins
Ca activationCa activation
myosin
actin
Force generationForce generation
function demands large conformational
changes;
myosin headmyosin head
troponin Ctroponin C
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Why EPR ?
•Orientation•Dynamics•Distances•2o structure
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HN
O
O
N
cysteine
IASL
N
cysteine
OO
O
N
MSL
N
O
O
O
InVSL
Labeling Cysteine ScanningLabeling Cysteine Scanning
Native cysteinesNative cysteines Cysteine scanningCysteine scanning
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Dipolar EPR: distancesDipolar EPR: distances
Non-interacting spins
Double labeled
Rabenstein & Shin, PNAS, 92 (1995)
sensitivity: 8-20 Å
nitroxide - nitroxide
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Distance : metal-nitroxideDistance : metal-nitroxide
Pulsed EPR
TT11
Time
Echo/2
Nitroxide (ms)
Gd3+
Dipolar interaction
(s)
Sensitivity: 10–50 Å
echo
inte
nsit
y
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DEER(Double Electron Electron
Resonance)
DEER(Double Electron Electron
Resonance)
/2 2
Echo
pump t
observe Dipolar interaction
Echo ModulationDEER Echo Modulation
350
400
450
500
550
600
650
0 200 400 600 800 1000 1200
Time(ns)
Ech
o am
plitu
de
Long Distance: 18 –50 Å
Sensitive to distance distribution
Model spectra
38 Å
25 Å
Milov, Jeschke
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Applications
Dipolar EPR
• myosin cleft closure
• myosin head interactions in smooth muscle
• troponin
• opening of K+ - channel
Site specific spin labelling
• structure of troponin I
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Actin binding cleft Actin binding cleft conformationconformation
416
537
A. Málnási-Csizmadia, C. Bagshaw, P. Connibear
force
Cleft closure associated with lever swing
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EPR distancesEPR distances
stateCw DEER
short long % long disp.
Acto.S1 12 26 15% 25 7
ADP 12 20 8% 23 7
AlF 12 20 7% 18 14
Apo 13 24 16% 24 8
Acto.S1
ADP
AlF
apo
Single
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0 0.2 0.4 0.6 0.8
time (us)
dipo
lar e
volu
tion
•distribution of distances•changing fraction of each
equilibrium of CLOSED and OPEN states shifts towards CLOSED in the presence of actin
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Wendt et al. (1999) Wahlstrom et al. (2003)
MD-MD RLC- RLC
Smooth muscle regulationSmooth muscle regulation
TaylorTaylor CremoCremo
•Hypothesis: heads stick together inhibiting ATPase
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RLC single cysteine mutantsRLC single cysteine mutants
TaylorTaylor CremoCremo
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EPR distances EPR distances
residue Cremo Taylor
38
59
84
108
23
•The measured distances are consistent with the Taylor model
•The N-terminal portion is further apart than either model
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Tung et. al, Protein Sci, 2000
47 Å
Vassylyev et al. PNAS, 1998
37 Å
Troponin: Collapse of central helix
Troponin: Collapse of central helix
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Troponin
Ca switch mechanism shown in isolated TnC but
NOT in ternary complex of TnI, TnC and TnT
Questions:
1. what is the structure of TnC in ICT complex ?
2. what are the Ca induced conformational changes
in ICT ?
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Collapse of TnC central helix
Collapse of TnC central helix
Spin labels: 12, 51, 89, 94
Gd3+: sites III & IV
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Isolated TnCIsolated TnC
Pulse
d EPR
X-ray
(5TNC)
NMR
(1AJ4)
TnC
9427 24 20
TnC
8928 29 28
TnC
1235 37 37
TnC
5147 45 43
Excellent agreement with X-ray and NMR
TnC in solution is extended
TnC51 T1 Enhancement
0
20
40
60
80
100
0 5 10 15 20 25
ms
Ech
o In
tens
ity
No Gd
With Gd
TnC89 T1 Enhancement
0
200
400
600
800
1000
1200
0 5 10 15 20 25
ms
Ech
o In
ten
sity
No Gd
With Gd
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Ternary complexTernary complex
Gd3+ to nitroxide distance
site TnC I.C.T change
TnC 94 27 29 +2
TnC 89 28 30 +2
TnC 51 47 38 -9
N- to C-domain distance decreases by 9 Ǻ central helix bends in a complex
37 Å
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N-domain: Homology model for Ca2+ switch in troponin
N-domain: Homology model for Ca2+ switch in troponin
0
5
10
15
20
25
30
35
40
An
gs
tro
m
DEER
MD
15-94 15-136 12-136
• distances consistent with the TnC based homology model
(assume no changes in the N-domain which senses Ca)
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C-domain of TnC C-domain of TnC
TnI 51
TnC 100
DEER cw-EPR
+Ca 19.5 18.1
apo 17.1 15.7
All distances are in (Ǻ)
TnI N-terminal helix moves v. little (2Å) with respect to TnC C-domain on Ca2+ binding.
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Conformational changes in a complex
Conformational changes in a complex
1. TnC is more compact in ternary complex than isolated TnC.
2. Calcium switch might well be same in troponin complex as in isolated TnC.
3. N-domain of TnI remains in proximity of C-domain of TnC.
Tn (+ Ca) =TnC Tn (- Ca)+
central helix bending N domain movement
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Opening of KOpening of K++ channel channel
Closed (x-ray) Open (homology)
Y. Li, E. PerozoY. Li, E. Perozo
Homology model is wrong.Homology model is wrong.
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Scatter = 6 Å
Fidelity of the EPR Fidelity of the EPR distancesdistances
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Molecular DynamicsMolecular DynamicsDistance
Spin-spin angle
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EPR v. X-ray/MD-MCEPR v. X-ray/MD-MC
Modelling the spin label decreases scatter = 3 Å
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EPR
Molecula
r
property
Signal
Power
saturation
Solvent
accessibilityAmplitude
Convention
al EPRMobility Splitting
Dipolar EPRSpin-spin
distance
Broadenin
g
Hubbell, 1989 “cysteine scanning” from 130-146
Site Directed Spin Labeling EPR
Site Directed Spin Labeling EPR
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Secondary structure determinationSecondary structure determination
power ½ (mW) ½
ampl
itu
de P1/2= 60 mW
P1/2= 20 mW
0 5 10 150
2
4
0 5 10 15 20
residue number0 5 10 15 20
residue number
P1/
2(O
2)/ P
1/2(
CR
OX
)
P1/
2(O
2)/ P
1/2(
CR
OX
)
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Computational modelsComputational models
-helix (x-ray)X-ray CS data, homology
model Vassylyev et al PNAS 95:4847 ‘98
-hairpin loop (nmr)Neutron scatteringTung et al Prot.Sci. 9:1312 ‘00
TnI inhibitory region
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hairpin SAS
residue no. (cardiac)
128 130 132 134 136 138 140
P
1/2(
NiE
DD
A-N
2)
0
100
200
300
400
500
skeletal94 96 98 100 102 104 106
Helix SAS
residue no. (cardiac)
128 130 132 134 136 138 140
P
1/2(
NiE
DD
A-N
2)
0
100
200
300
400
500
skeletal94 96 98 100 102 104 106
130-138 region is a helix130-138 region is a helix
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138-146 region138-146 regionSolvent accessibility
residue no. (cardiac)
138 140 142 144 146 148
P
1/2(
NiE
DD
A-N
2)
100
200
300
400
500
skeletal104 106 108 110 112 114
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130.plt;
131.plt;
132.plt;
133.plt;
134.plt;
135.plt;
136.plt;
137.plt;
Identifying the interface between subunits
Identifying the interface between subunits
130-136TnT imprint
130
131
132
133
134
135
136
137
Ternary: TnI mutants
Binary/ternary “difference”
map
200
0.006
ICT/IC
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Summary
1. Dipolar EPR excellent for 10-20 A
2. Pulsed EPR extends the range to 20-50 A
3. “Easy” protein chemistry
4. Large macromolecular complexes
5. Determination of secondary structure.
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The LabThe Lab
•Hua Liang
•Song Likai
•Clement
Rouviere
•Louise Brown
•Ken Sale
•Hua Liang
•Song Likai
•Clement
Rouviere
•Louise Brown
•Ken Sale
Collaborators
Clive Bagshaw ~ U. Leicester
A.Málnási-Csizmadia ~ Eötvös U.
E. Perozo ~ U. Virginia
Collaborators
Clive Bagshaw ~ U. Leicester
A.Málnási-Csizmadia ~ Eötvös U.
E. Perozo ~ U. Virginia