Florida State University, National High Magnetic Fields Laboratory Piotr Fajer Conformational Changes Associated with Muscle Activation and Force Generation by Pulsed EPR Methods
Dec 23, 2015
Florida State University,
National High Magnetic
Fields Laboratory
Piotr Fajer
Conformational Changes Associated with Muscle Activation
and Force Generation by Pulsed EPR Methods
Motor proteinsMotor proteinsMotor proteinsMotor proteins
Ca activationCa activation
myosin
actin
Force generationForce generation
function demands large conformational
changes;
myosin headmyosin head
troponin Ctroponin C
Why EPR ?
•Orientation•Dynamics•Distances•2o structure
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
Dipolar EPR: distancesDipolar EPR: distances
Non-interacting spins
Double labeled
Rabenstein & Shin, PNAS, 92 (1995)
sensitivity: 8-20 Å
nitroxide - nitroxide
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
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
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
Actin binding cleft Actin binding cleft conformationconformation
416
537
A. Málnási-Csizmadia, C. Bagshaw, P. Connibear
force
Cleft closure associated with lever swing
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
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
RLC single cysteine mutantsRLC single cysteine mutants
TaylorTaylor CremoCremo
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
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
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 ?
Collapse of TnC central helix
Collapse of TnC central helix
Spin labels: 12, 51, 89, 94
Gd3+: sites III & IV
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
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
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)
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.
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
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.
Scatter = 6 Å
Fidelity of the EPR Fidelity of the EPR distancesdistances
Molecular DynamicsMolecular DynamicsDistance
Spin-spin angle
EPR v. X-ray/MD-MCEPR v. X-ray/MD-MC
Modelling the spin label decreases scatter = 3 Å
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
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
)
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
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
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
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
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.
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