conformational changes associated with muscle activation and force generation by pulsed epr methods
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Florida State University, National High Magnetic Fields Laboratory. Conformational Changes Associated with Muscle Activation and Force Generation by Pulsed EPR Methods. Piotr Fajer. myosin head. Motor proteins. Ca activation. actin. troponin C. myosin. Force generation.

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motor proteins

myosin head

Motor proteins

Ca activation

actin

troponin C

myosin

Force generation

function demands large conformational changes;

why epr
Why EPR ?
  • Orientation
  • Dynamics
  • Distances
  • 2o structure
labeling cysteine scanning

O

O

N

O

InVSL

cysteine

O

cysteine

N

O

H

N

O

N

N

O

O

IASL

MSL

Labeling Cysteine Scanning

Cysteine scanning

Native cysteines

dipolar epr distances

nitroxide - nitroxide

Dipolar EPR: distances

Rabenstein & Shin, PNAS, 92 (1995)

Non-interacting spins

Double labeled

sensitivity: 8-20 Å

distance metal nitroxide

Echo

/2

Nitroxide (ms)

Dipolar interaction

Gd3+

(s)

Distance : metal-nitroxide

Pulsed EPR

T1

echo intensity

Time

Sensitivity: 10–50 Å

deer double electron electron resonance
DEER(Double Electron Electron Resonance)

Echo

/2

Dipolar interaction

nobserve

t2

t1

t1

npump

t

Echo Modulation

Milov, Jeschke

Model spectra

Long Distance: 18 –50 Å

Sensitive to distance distribution

25 Å

38 Å

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

Applications
actin binding cleft conformation
Actin binding cleft conformation

416

537

A. Málnási-Csizmadia, C. Bagshaw, P. Connibear

force

Cleft closure associated with lever swing

epr distances
EPR distances
  • distribution of distances
  • changing fraction of each 
  • equilibrium of CLOSED and OPEN states shifts towards CLOSED in the presence of actin
smooth muscle regulation
Smooth muscle regulation

RLC- RLC

MD-MD

Wendt et al. (1999)

Wahlstrom et al. (2003)

Taylor

Cremo

  • Hypothesis: heads stick together inhibiting ATPase
epr distances1
EPR distances
  • The measured distances are consistent with the Taylor model
  • The N-terminal portion is further apart than either model
troponin collapse of central helix
Troponin: Collapse of central helix

47 Å

37 Å

Vassylyev et al. PNAS, 1998

Tung et. al, Protein Sci, 2000

troponin
Ca switch mechanism shown in isolated TnC but NOT in ternary complex of TnI, TnC and TnT

Questions:

what is the structure of TnC in ICT complex ?

what are the Ca induced conformational changes in ICT ?

Troponin
collapse of tnc central helix
Collapse of TnC central helix

Spin labels: 12, 51, 89, 94

Gd3+: sites III & IV

isolated tnc
Isolated TnC

Excellent agreement with X-ray and NMR

TnC in solution is extended

ternary complex
Ternary complex

37 Å

N- to C-domain distance decreases by 9 Ǻ central helix bends in a complex

n domain homology model for ca 2 switch in troponin

15-94 15-136 12-136

N-domain: Homology model for Ca2+ switch in troponin

(assume no changes in the N-domain which senses Ca)

  • distances consistent with the TnC based homology model
c domain of tnc
C-domain of TnC

TnI 51

TnC 100

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
TnC is more compact in ternary complex than isolated TnC.

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.

N domain movement

central helix bending

Tn (+ Ca)

Tn (- Ca)

TnC

+

=

Conformational changes in a complex
opening of k channel
Opening of K+ channel

Y. Li, E. Perozo

Closed (x-ray)

Open (homology)

Homology model is wrong.

molecular dynamics
Molecular Dynamics

Distance

Spin-spin angle

epr v x ray md mc
EPR v. X-ray/MD-MC

Modelling the spin label decreases scatter = 3 Å

site directed spin labeling epr
Site Directed Spin Labeling EPR

Hubbell, 1989

“cysteine scanning” from 130-146

secondary structure determination

P1/2(O2)/ P1/2(CROX)

P1/2(O2)/ P1/2(CROX)

Secondary structure determination

4

P1/2= 60 mW

amplitude

2

P1/2= 20 mW

0

0

5

10

15

power ½ (mW) ½

computational models
Computational models

TnI inhibitory region

-helix (x-ray)

X-ray CS data, homology model

Vassylyev et al PNAS 95:4847 ‘98

-hairpin loop (nmr)

Neutron scattering

Tung et al Prot.Sci. 9:1312 ‘00

identifying the interface between subunits
Identifying the interface between subunits

Binary/ternary  “difference” map

130

131

132

130-136

TnT imprint

133

134

135

136

200

137

tICT/tIC

Ternary: TnI mutants

0.006

summary
Dipolar EPR excellent for 10-20 A

Pulsed EPR extends the range to 20-50 A

“Easy” protein chemistry

Large macromolecular complexes

Determination of secondary structure.

Summary
the lab
Hua Liang

Song Likai

Clement Rouviere

Louise Brown

Ken Sale

The Lab
  • Collaborators
  • Clive Bagshaw ~ U. Leicester
  • Málnási-Csizmadia ~ Eötvös U.
  • E. Perozo ~ U. Virginia
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