Lisa A. Compton

1
Structure of the inhibitory region of troponin by
site directed spin labeling electron paramagnetic
resonance Louise J. Brown, Ken L. Sale, Ron
Hills, Clement Rouviere, Likai Song, Xiaojun
Zhang, and Piotr G. Fajer PNAS, October 2002,
99(20) 12765-12770

• Lisa A. Compton
• April 22, 2004

2
Outline

• Introduction
• Methods
• Figures from Paper
• Conclusion

3
Introduction

• Troponin (Tn) complex TnT, TnI, TnC
• Inhibitory region of TnI
• Maeda and friends a-helix
• Trewhella and friends flexible b-hairpin
• Fajer and friends to solve the puzzle

4
Methods

• Troponin Subunits
• cTnT extracted from bovine hearts
• cTnI expressed in pET3d vector, mouse
• sTnC expressed in pET3d vector, chicken
• Mutations
• Mutated native Cys to Ser cysteine-less
• Mutated sites of interest to cysteines

5
Methods

• Purified proteins were labeled with MTSSL
  (1-oxyl-2,2,5,5-tetramethyl- 3-pyrroline-3-(methyl
  )methanethiosulfonate spin label)
• Reconstitution of the binary or ternary complexes
• Checked reconstitution by gel electrophoresis and
  function by in vitro motility

6
Methods

• EPR spectra were recorded on a Bruker EMX 9 GHz
  spectrometer at 20C
• Oxygen was removed by purging with nitrogen gas
• Nitroxide accessibility to NiEDDA (water soluble
  relaxant) by the power saturation technique
• Helical periodicity was measured as the
  contribution of the components between 80 and
  120 of the power Fourier transform of the
  accessibility data (over a seven residue window)

7
Methods

• Spin-spin distances were measured from EPR
  dipolar interactions of doubly labeled samples
  (that were frozen) using a microwave power of 10
  mW, modulation amplitude of 1 G, and a scan range
  of 180 G
• Double electron-electron resonance (DEER) was
  performed on a Bruker 680 Fourier transform
  spectrometer using a 4 pulse sequence
  (90-t1-180-t2)

8
Profile of Solvent Accessibility of TnI in the
Binary Complex
No discernible trend between these residues
9
Accessibility Profile of the Inhibitory Region in
the Ternary Tn Complex
Follow an a-helical pattern
Do not show any discernable trend
10
Distances Between TnC and TnI Determined by
Dipolar EPR in the Binary Complex
Inhibitory region of TnI is not making extended
contact with the central helix of TnC (as
originally thought)
11
Spin-Spin Distances Within the Inhibitory Region
of TnI as Determined by Dipolar EPR in the Binary
Complex
Region 140-145 is most likely an a-helix and
145-149 is closer to an a-helix than a b-sheet
12
Conclusion

• The inhibitory region of TnI is an a-helix