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Florida State University, National High Magnetic Fields Laboratory

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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Florida State University, National High Magnetic Fields Laboratory

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  1. Florida State University, National High Magnetic Fields Laboratory Conformational Changes Associated with Muscle Activation and Force Generation by Pulsed EPR Methods Piotr Fajer

  2. myosin head Motor proteins Ca activation actin troponin C myosin Force generation function demands large conformational changes;

  3. Why EPR ? • Orientation • Dynamics • Distances • 2o structure

  4. 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

  5. nitroxide - nitroxide Dipolar EPR: distances Rabenstein & Shin, PNAS, 92 (1995) Non-interacting spins Double labeled sensitivity: 8-20 Å

  6. Echo  /2  Nitroxide (ms) Dipolar interaction Gd3+ (s) Distance : metal-nitroxide Pulsed EPR T1 echo intensity Time Sensitivity: 10–50 Å

  7. 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

  8. 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

  9. Actin binding cleft conformation 416 537 A. Málnási-Csizmadia, C. Bagshaw, P. Connibear force Cleft closure associated with lever swing

  10. EPR distances • distribution of distances • changing fraction of each  • equilibrium of CLOSED and OPEN states shifts towards CLOSED in the presence of actin

  11. Smooth muscle regulation RLC- RLC MD-MD Wendt et al. (1999) Wahlstrom et al. (2003) Taylor Cremo • Hypothesis: heads stick together inhibiting ATPase

  12. RLC single cysteine mutants Taylor Cremo

  13. EPR distances • The measured distances are consistent with the Taylor model • The N-terminal portion is further apart than either model

  14. Troponin: Collapse of central helix 47 Å 37 Å Vassylyev et al. PNAS, 1998 Tung et. al, Protein Sci, 2000

  15. 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

  16. Collapse of TnC central helix Spin labels: 12, 51, 89, 94 Gd3+: sites III & IV

  17. Isolated TnC Excellent agreement with X-ray and NMR TnC in solution is extended

  18. Ternary complex 37 Å N- to C-domain distance decreases by 9 Ǻ central helix bends in a complex

  19. 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

  20. 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.

  21. 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

  22. Opening of K+ channel Y. Li, E. Perozo Closed (x-ray) Open (homology) Homology model is wrong.

  23. Fidelity of the EPR distances Scatter = 6 Å

  24. Molecular Dynamics Distance Spin-spin angle

  25. EPR v. X-ray/MD-MC Modelling the spin label decreases scatter = 3 Å

  26. Site Directed Spin Labeling EPR Hubbell, 1989 “cysteine scanning” from 130-146

  27. 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

  28. 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

  29. 130-138 region is a helix

  30. 138-146 region

  31. 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

  32. 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

  33. 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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