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 requirement 2- nature of the environment 3- Dynamics and time scales

2. Functional Dynamics of Proteins

5. N O CH 2 SSCH 3 O O S C NH CH C NH CH C O O O NH 3 + C NH CH C NH CH C O O O NH 3 + SH C NH CH C NH CH C O O O N H 3 + S N O Side Chain R1 Site-Directed Spin Labeling

6. 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

7. Electron Paramagnetic Resonance Electron Spin Resonance Mn +2 OH. N O R 1 R 2. Free Radicals Ascorbate Hydroxyl Tocopherol etc. Transition Metals All that have unpaired electrons Spin Labels R 1 & R 2 chosen to provide specificity

8. Spin ½ Electron in an External Field Electron Spins Precess @ the Larmor Frequency about the External Field  e = 1.76e 7 rad/sec/G

9. Approximate Energy Levels for a 14 N Nitroxide Spin Label; S =½, I=1 In general, EPR gives information on the # and nuclear spin state of nearby nuclei – coordination sphere of metal centers in in metalloproteins

10. Energy level Diagram for a 15 N Nitroxide S=½; I=½

11. Block Diagram of EPR Spectrometer Solids Liquids Gases High S/N Most EPR done @ X-band 9-10 GHz 3 kG Field

12. Nitroxide Reference Frame H res = [h /  e g eff  )] + m i A eff (  ) g eff = g xx sin 2  cos 2  g yy sin 2  sin 2  + g zz cos 2  A eff = [A xx 2 sin 2  cos 2  + A yy 2 sin 2  sin 2  + A zz 2 cos 2  ] ½ g xx = 2.0086 g yy = 2.0056 g zz = 2.0022 A xx = 7 G A yy = 6 G A zz = 34 G

13. Variation in the EPR Spectrum for a Single Nitroxide Single Crystal - x Single Crystal - y Single Crystal - z Polycrystalline Crystal Dissolved in Water

14. Two-Site Exchange Model Effects of Motion on EPR Spectra

15. EPR Spectra as a Function of Rotational Correlation Time;  r = 1/6D r Nitroxide in H 2 0 Small Protein - EGF Large Protein – 1 MDa  r = 4  r h 3 / 3kT

16. Electron Spin-Spin Relaxation - Accessibility Nitroxide – slow relaxation T 1e ’s of isolated Nitroxide spin labels are on the order of a few  sec’s at physiological temperatures

17. Electron Spin-Spin Relaxation - Accessibility Nitroxide – slow relaxation Nicklel Complexes – fast relaxation Other Transition Metals and O 2