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

2. 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

3. 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

4. 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

5. 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 = nsec 4
tr = nsec
tr = nsec
tr = nsec
tr = nsec
Biophysical Tools '02 - EPR
12/3/2018

6. 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

7. 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

8. 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

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

10. 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

11. 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