1. Lisa A. Compton April 22, 2004 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

2. Outline Introduction Methods Figures from Paper Conclusion

3. Introduction Troponin (Tn) complex – TnT, TnI, TnC Inhibitory region of TnI –Maeda and friends –  -helix –Trewhella and friends – flexible  -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 20’C 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  -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  -helix than a  -sheet

12. Conclusion The inhibitory region of TnI is an  -helix