Conformational changes associated with muscle activation and force generation by pulsed epr methods
Download
1 / 33

Florida State University, National High Magnetic Fields Laboratory - PowerPoint PPT Presentation


  • 76 Views
  • Uploaded on

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.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Florida State University, National High Magnetic Fields Laboratory ' - vinaya


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
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 Laboratory

Motor proteins

Ca activation

actin

troponin C

myosin

Force generation

function demands large conformational changes;


Why epr
Why EPR ? Laboratory

  • Orientation

  • Dynamics

  • Distances

  • 2o structure


Labeling cysteine scanning

O Laboratory

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 Laboratory

Dipolar EPR: distances

Rabenstein & Shin, PNAS, 92 (1995)

Non-interacting spins

Double labeled

sensitivity: 8-20 Å


Distance metal nitroxide

Echo Laboratory

/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 Laboratory (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 Laboratory

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 Laboratory

416

537

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

force

Cleft closure associated with lever swing


Epr distances
EPR distances Laboratory

  • 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 Laboratory

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 Laboratory

Taylor

Cremo


Epr distances1
EPR distances Laboratory

  • 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 Laboratory

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 ternary complex of TnI, TnC and TnTcentral helix

Spin labels: 12, 51, 89, 94

Gd3+: sites III & IV


Isolated tnc
Isolated TnC ternary complex of TnI, TnC and TnT

Excellent agreement with X-ray and NMR

TnC in solution is extended


Ternary complex
Ternary complex ternary complex of TnI, TnC and TnT

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 ternary complex of TnI, TnC and TnT

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 ternary complex of TnI, TnC and TnT 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. ternary complex of TnI, TnC and TnT

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 ternary complex of TnI, TnC and TnT+ channel

Y. Li, E. Perozo

Closed (x-ray)

Open (homology)

Homology model is wrong.


Fidelity of the epr distances
Fidelity of the EPR distances ternary complex of TnI, TnC and TnT

Scatter = 6 Å


Molecular dynamics
Molecular Dynamics ternary complex of TnI, TnC and TnT

Distance

Spin-spin angle


Epr v x ray md mc
EPR v. X-ray/MD-MC ternary complex of TnI, TnC and TnT

Modelling the spin label decreases scatter = 3 Å


Site directed spin labeling epr
Site Directed Spin Labeling EPR ternary complex of TnI, TnC and TnT

Hubbell, 1989

“cysteine scanning” from 130-146


Secondary structure determination

P ternary complex of TnI, TnC and TnT1/2(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 ternary complex of TnI, TnC and TnT

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 ternary complex of TnI, TnC and TnT


138 146 region
138-146 region ternary complex of TnI, TnC and TnT


Identifying the interface between subunits
Identifying the interface between subunits ternary complex of TnI, TnC and TnT

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 ternary complex of TnI, TnC and TnT

Pulsed EPR extends the range to 20-50 A

“Easy” protein chemistry

Large macromolecular complexes

Determination of secondary structure.

Summary


The lab

Hua Liang ternary complex of TnI, TnC and TnT

Song Likai

Clement Rouviere

Louise Brown

Ken Sale

The Lab

  • Collaborators

  • Clive Bagshaw ~ U. Leicester

  • Málnási-Csizmadia ~ Eötvös U.

  • E. Perozo ~ U. Virginia


ad