Epr 1

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 requirement2- nature of the environment3- Dynamics and time scales

Functional Dynamics of Proteins

NO

CH2 S S CH3

O

OMethanethiosulfonate

spin label

Site-directed mutagenesis

S

C NHCH

CNH

CHC

O

O

O

NH3+

C NHCH

CNH

CHC

O

O

O

NH3+SH

C NHCH

CNH

CHC

O

O

O

NH3+

S

N O

Side Chain R1

Site-Directed Spin Labeling

Spectral parameter Protein structure

Probe spectroscopy

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

Electron Paramagnetic ResonanceElectron Spin Resonance

Mn+2

OH. N

O

R1 R2

.

Free RadicalsAscorbateHydroxylTocopheroletc.

Transition MetalsAll that haveunpaired electrons

Spin LabelsR1 & R2 chosen to provide specificity

Spin ½ Electron in an External Field

Electron Spins Precess@ the Larmor Frequencyabout the External Field

γe = 1.76e7 rad/sec/G

Approximate Energy Levels for a 14N Nitroxide Spin Label; S =½, I=1

In general, EPR givesinformation on the #and nuclear spin stateof nearby nuclei –coordination sphere ofmetal centers in in metalloproteins

Energy level Diagram for a 15N NitroxideS=½; I=½

Block Diagram of EPR Spectrometer

SolidsLiquidsGasesHigh S/N

Most EPRdone @X-band9-10 GHz3 kG Field

Nitroxide Reference Frame

Hres = [hν/βegeff(θ,φ)] + miAeff(θ,φ)

geff = gxxsin2θcos2φ + gyysin2θsin2φ + gzzcos2θ

Aeff = [Axx2sin2θcos2φ + Ayy

2sin2θsin2φ + Azz2cos2θ] ½

gxx = 2.0086gyy = 2.0056gzz = 2.0022Axx = 7 GAyy = 6 GAzz = 34 G

Variation in the EPR Spectrum for a Single Nitroxide

Single Crystal - x

Single Crystal - y

Single Crystal - z

Polycrystalline

Crystal Dissolvedin Water

Two-Site Exchange ModelEffects of Motion on EPR Spectra

EPR Spectra as a Function of RotationalCorrelation Time; τr = 1/6Dr

Nitroxide in H20

Small Protein - EGF

Large Protein – 1 MDa

τr = 4πηrh3/ 3kT

Electron Spin-Spin Relaxation - Accessibility

Nitroxide – slow relaxation

T1e’s of isolated Nitroxide spin labels are on the order of afew µsec’s at physiological temperatures

Electron Spin-Spin Relaxation - Accessibility

Nitroxide – slow relaxation

Nicklel Complexes – fast relaxationOther Transition Metals and O2