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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Conformational changes associated with muscle activation and force generation by pulsed epr methods

Florida State University, National High Magnetic Fields Laboratory

Conformational Changes Associated with Muscle Activation and Force Generation by Pulsed EPR Methods

Piotr Fajer


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


Rlc single cysteine mutants

RLC single cysteine mutants

Taylor

Cremo


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.


Fidelity of the epr distances

Fidelity of the EPR distances

Scatter = 6 Å


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


130 138 region is a helix

130-138 region is a helix


138 146 region

138-146 region


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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