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

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

3. Why EPR ? Orientation Dynamics Distances 2 o structure

4. HN O O N cysteine IASL N cysteine O O O N MSL N O O O InVSL Labeling Cysteine Scanning Native cysteines Cysteine scanning

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

6. Distance : metal-nitroxide Pulsed EPR T1T1T1T1 Time Echo  /2  Nitroxide (ms) Gd 3+ Dipolar interaction (  s) Sensitivity: 10–50 Å echo intensity

7. DEER (Double Electron Electron Resonance)   /2  22 Echo  pump t observe  Dipolar interaction Echo Modulation Long Distance: 18 –50 Å Sensitive to distance distribution Model spectra 38 Å 25 Å Milov, Jeschke

8. 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

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

10. EPR distances state CwDEER shortlong% disp. Acto.S1122615%257 ADP12208%237 AlF12207%1814 Apo132416%248 distribution of distances changing fraction of each  equilibrium of CLOSED and OPEN states shifts towards CLOSED in the presence of actin

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

12. RLC single cysteine mutants TaylorCremo

13. EPR distances residueCremoTaylor 38 59 84  108 23  The measured distances are consistent with the Taylor model The N-terminal portion is further apart than either model

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

15. Troponin Ca switch mechanism shown in isolated TnC but NOT in ternary complex of TnI, TnC and TnT Questions: 1.what is the structure of TnC in ICT complex ? 2.what are the Ca induced conformational changes in ICT ?

16. Collapse of TnC central helix Spin labels: 12, 51, 89, 94 Gd 3+ : sites III & IV

17. Isolated TnC Pulsed EPR X-ray (5TNC) NMR (1AJ4) TnC 94272420 TnC 8928 2928 TnC 123537 TnC 51474543 Excellent agreement with X-ray and NMR TnC in solution is extended

18. Ternary complex Gd 3+ to nitroxide distance siteTnCI.C.T change TnC 942729+2 TnC 892830+2 TnC 51 4738-9 N- to C-domain distance decreases by 9 Ǻ  central helix bends in a complex 37 Å

19. N-domain: Homology model for Ca 2+ switch in troponin 15-94 15-136 12-136 distances consistent with the TnC based homology model (assume no changes in the N-domain which senses Ca)

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 Ca 2+ binding.

21. Conformational changes in a complex 1.TnC is more compact in ternary complex than isolated TnC. 2.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. Tn (+ Ca) = TnC Tn (- Ca) + central helix bending N domain movement

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

23. Scatter = 6 Å Fidelity of the EPR distances

24. Molecular Dynamics Distance Spin-spin angle

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

26. EPR Molecular property Signal Power saturation Solvent accessibility Amplitude Conventional EPR MobilitySplitting Dipolar EPR Spin-spin distance Broadening Hubbell, 1989 “cysteine scanning” from 130-146 Site Directed Spin Labeling EPR

27. Secondary structure determination power ½ (mW) ½ amplitude P 1/2 = 60 mW P 1/2 = 20 mW 051015 0 2 4 P 1/2 (O 2 )/ P 1/2 (CROX)

28. Computational models  -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 TnI inhibitory region

29. 130-138 region is a helix

30. 138-146 region

31. Identifying the interface between subunits 130-136 TnT imprint 130 131 132 133 134 135 136 137 Ternary: TnI mutants Binary/ternary  “difference” map 200 0.006  ICT /  IC

32. Summary 1. Dipolar EPR excellent for 10-20 A 2. Pulsed EPR extends the range to 20-50 A 3. “Easy” protein chemistry 4. Large macromolecular complexes 5. Determination of secondary structure.

33. The Lab Hua Liang Song Likai Clement Rouviere Louise Brown Ken Sale Collaborators Clive Bagshaw ~ U. Leicester A. A.Málnási-Csizmadia ~ Eötvös U. E. Perozo ~ U. Virginia