Florida State University, National High Magnetic Fields Laboratory Piotr Fajer Conformational Changes Associated with Muscle Activation and Force Generation by Pulsed EPR Methods
Motor proteins Ca activation myosin actin Force generation function demands large conformational changes; myosin head troponin C
Why EPR ? Orientation Dynamics Distances 2 o structure
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
Dipolar EPR: distances Non-interacting spins Double labeled Rabenstein & Shin, PNAS, 92 (1995) sensitivity: 8-20 Å nitroxide - nitroxide
Distance : metal-nitroxide Pulsed EPR T1T1T1T1 Time Echo /2 Nitroxide (ms) Gd 3+ Dipolar interaction ( s) Sensitivity: 10–50 Å echo intensity
DEER (Double Electron Electron Resonance) /2 22 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
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
Actin binding cleft conformation A. Málnási-Csizmadia, C. Bagshaw, P. Connibear force Cleft closure associated with lever swing
EPR distances state CwDEER shortlong% disp. Acto.S %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
Wendt et al. (1999)Wahlstrom et al. (2003) MD-MD RLC- RLC Smooth muscle regulationTaylorCremo Hypothesis: heads stick together inhibiting ATPase
RLC single cysteine mutants TaylorCremo
EPR distances residueCremoTaylor The measured distances are consistent with the Taylor model The N-terminal portion is further apart than either model
Tung et. al, Protein Sci, Å Vassylyev et al. PNAS, Å Troponin: Collapse of central helix
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 ?
Collapse of TnC central helix Spin labels: 12, 51, 89, 94 Gd 3+ : sites III & IV
Isolated TnC Pulsed EPR X-ray (5TNC) NMR (1AJ4) TnC TnC TnC TnC Excellent agreement with X-ray and NMR TnC in solution is extended
Ternary complex Gd 3+ to nitroxide distance siteTnCI.C.T change TnC TnC TnC N- to C-domain distance decreases by 9 Ǻ central helix bends in a complex 37 Å
N-domain: Homology model for Ca 2+ switch in troponin distances consistent with the TnC based homology model (assume no changes in the N-domain which senses Ca)
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.
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
Opening of K + channel Closed (x-ray)Open (homology) Y. Li, E. Perozo Homology model is wrong.
Scatter = 6 Å Fidelity of the EPR distances
Molecular Dynamics Distance Spin-spin angle
EPR v. X-ray/MD-MC Modelling the spin label decreases scatter = 3 Å
EPR Molecular property Signal Power saturation Solvent accessibility Amplitude Conventional EPR MobilitySplitting Dipolar EPR Spin-spin distance Broadening Hubbell, 1989 “cysteine scanning” from Site Directed Spin Labeling EPR
Secondary structure determination power ½ (mW) ½ amplitude P 1/2 = 60 mW P 1/2 = 20 mW P 1/2 (O 2 )/ P 1/2 (CROX)
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
region is a helix
region
Identifying the interface between subunits TnT imprint Ternary: TnI mutants Binary/ternary “difference” map ICT / IC
Summary 1. Dipolar EPR excellent for A 2. Pulsed EPR extends the range to A 3. “Easy” protein chemistry 4. Large macromolecular complexes 5. Determination of secondary structure.
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