Electron Paramagnetic Resonance

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Tools of the Trade
1- Atomic resolution X-ray crystallography 2-
NMR spectroscopy 3- de novo Modeling and
structure determination, Homology modeling 4-
CryoEM Why do we needs probes ????? Issues
1- sample requirement 2- nature of the
environment 3- Dynamics and time scales
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Functional Dynamics of Proteins
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Site-Directed Spin Labeling
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Probe spectroscopy
Spectral parameter Protein
structure
Biggest challenge What is the transfer
function? Examples Uncertainty principle of
chemical modification Implications limited
resolution both on the static and dynamic
levels Functional analysis of labeled mutants
is a REQUIREMENT
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Electron Paramagnetic ResonanceElectron Spin
Resonance
R1 R2
OH.
N
.
Mn2
O
Free Radicals Ascorbate Hydroxyl Tocopherol etc.
Transition Metals All that have unpaired electrons
Spin Labels R1 R2 chosen to provide specificity
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Spin ½ Electron in an External Field
Electron Spins Precess _at_ the Larmor
Frequency about the External Field ge 1.76e7
rad/sec/G
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Approximate Energy Levels for a 14N Nitroxide
Spin Label S ½, I1
In general, EPR gives information on the and
nuclear spin state of nearby nuclei
coordination sphere of metal centers in in
metalloproteins
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Energy level Diagram for a 15N Nitroxide S½ I½
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Block Diagram of EPR Spectrometer
Solids Liquids Gases High S/N Most EPR done
_at_ X-band 9-10 GHz 3 kG Field
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Nitroxide Reference Frame
gxx 2.0086 gyy 2.0056 gzz 2.0022 Axx 7
G Ayy 6 G Azz 34 G
Hres hn/begeff(q,f) miAeff(q,f)
geff gxxsin2qcos2f gyysin2qsin2f gzzcos2q
Aeff Axx2sin2qcos2f Ayy2sin2qsin2f
Azz2cos2q ½
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Variation in the EPR Spectrum for a Single
Nitroxide
Single Crystal - x
Single Crystal - y
Single Crystal - z
Polycrystalline
Crystal Dissolved in Water
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Two-Site Exchange Model Effects of Motion on EPR
Spectra
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EPR Spectra as a Function of Rotational Correlatio
n Time tr 1/6Dr
tr 4phrh3/ 3kT
Small Protein - EGF
Nitroxide in H20
Large Protein 1 MDa
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Electron Spin-Spin Relaxation - Accessibility
Nitroxide slow relaxation
T1es of isolated Nitroxide spin labels are on
the order of a few msecs at physiological
temperatures
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Electron Spin-Spin Relaxation - Accessibility
Nitroxide slow relaxation
Nicklel Complexes fast relaxation Other
Transition Metals and O2