Electron Paramagnetic Resonance Biophysical Tools '02 - EPR 12/3/2018

Biophysical Tools '02 - EPR Physical principle Hyperfine coupling +½ -½ -1 +1 I S Ho E hn Same as in NMR: spinning charge  magnetic dipole  interaction with external magnetic field Transitions governed by selection rules: DM = 1 Hamiltonian: Hyperfine coupling Nuclear spin can couple to the electron spin and split further the levels. CW is usual but Fourier Transforms exist too. First derivative signals Numerous frequencies: X-band (9 GHz), K-band (18 GHz), Q-band (35 GHz), W-band (90 GHz) Biophysical Tools '02 - EPR 12/3/2018

Biophysical Tools '02 - EPR Instrument N O M S L H 3 C T amplifier klystron detector diode cysteine cys707 ELC cys177 RLC cys125 RLC cys154 resonator magnet Biophysical Tools '02 - EPR 12/3/2018

Anisotropy and orientation • average angle Ÿ disorder H o || x y z N O disorder width both the g-factor and the hyperfine couplings are tensors  different values in different directions average angle splitting Biophysical Tools '02 - EPR 12/3/2018

Biophysical Tools '02 - EPR Rotational mobility tr = 20 nsec tr = 200 msec tr = 10 nsec tr = 23 msec Tumbling molecule averages the anisotropy of g- and A- tensors if it is fast enough (1/tc  Dwanisotropy) tr = 4 msec tr = 0.001 nsec 4 tr = 0.001 nsec tr = 0.001 nsec tr = 0.001 nsec tr = 0.001 nsec Biophysical Tools '02 - EPR 12/3/2018

Head Orientation and Mobility in ATPase cycle force / movement sequential transitions from disordered-mobile myosin heads to ordered-immobile heads drives the generation of force Biophysical Tools '02 - EPR 12/3/2018

Site Directed Spin Labeling EPR Hubbell, 1989 Spectroscopy Molecular property Signal Power saturation Solvent accessibility Amplitude Conventional EPR Mobility Splitting Dipolar EPR Spin-spin distance Broadening AIM/METHODS SLIDE -aim: secondary structure of the inhibitory region and its location within the complex -new novel approach to looking at the intact complex in solution. Pionerred by Hubbell -methods we used involved a combination of cysteine substitution mutagenesis or scanning, followed by the attachment of the the nitroxide spin label to the cysteines and then the measurement of several spectral properties of the spin label by EPR spectroscopy. These few parameters can provide a wealth of information on the environment in the protein. Solvent accessibility to the aqueous EPR relaxant NiEDDA as measured by power saturation. This relies on having an area of unequal solvation of a protein surface so as to measure the side chain environments which in turn can provide accessibility patterns that can identify secondary structure through a scanned region. “periodic function of sequence position Mobility of the side chain: is deduced from the spectral line shape and is measured by the spin label averaging of the hyperfine anisotropy or splitting?? Is a measure of sidechains involved in tertiary or quaternary interactions as in our case where the side chain mobility becomes restricted. Both models predicted a solvent inaccessible interface between the inhibitory region and TnC central helix that would be expected to allow us to distinguish between the two types of secondary structure by the power saturation technique and also by sidechain mobility (iii) Distance between two spin labels is also deduced from the spectral line shape for distances in the range of 8-25A We also selected distance pairs between the inhibitory region and central helix of TnC that were predicted by both models to be within the 20A limit of the dipolar EPR technique and that would also help us distinguish between models. “cysteine scanning” from 130-146

Secondary structure determination P1/2(O2)/ P1/2(CROX) power ½ (mW) ½ amplitude P1/2= 60 mW P1/2= 20 mW 5 10 15 2 4 Biophysical Tools '02 - EPR 12/3/2018

Dipolar EPR: distances Rabenstein & Shin, PNAS, 92 (1995) Range: 8-20 Å Sample spectra Model Shin’s curve Non-interacting spins Double labeled

Biophysical Tools '02 - EPR Spin trapping Biological radicals are very, very short lived - diffcult to detect Nitrones capture radicals changing into nitroxides with characteristic spectrum Biophysical Tools '02 - EPR 12/3/2018

Biophysical Tools '02 - EPR Transition metals d-orbitals of transition metals the only method looking at the metal catalysts Biophysical Tools '02 - EPR 12/3/2018