1. The document discusses several techniques for determining protein structure like X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. 2. Site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy are introduced as techniques to study the functional dynamics and interactions within proteins over different time scales. 3. EPR provides information about the local environment and dynamics of spin labels attached to specific sites within a protein, but the technique has limitations in resolution due to the nature of chemical modification and spin label dynamics.

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. Site-Directed Spin Labeling N O C H 2 S S C H 3 O O S C N H C H C N H C H C O O O N H 3 + C N H C H C N H C H C O O O N H 3 + S H C N H C H C N H C H C O O O N H 3 + S N O Side Chain R1

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. E lectron P aramagnetic R esonance E lectron S pin R esonance 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