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