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Tools of the Trade

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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Tools of the Trade

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  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

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

  4. 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

  5. Electron ParamagneticResonanceElectron Spin Resonance R1 R2 N . OH. Mn+2 O Free Radicals Ascorbate Hydroxyl Tocopherol etc. Transition Metals All that have unpaired electrons Spin Labels R1 & R2 chosen to provide specificity

  6. Spin ½ Electron in an External Field Electron Spins Precess @ the Larmor Frequency about the External Field ge = 1.76e7 rad/sec/G

  7. Approximate Energy Levels for a 14N 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

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

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

  10. 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

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

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

  13. EPR Spectra as a Function of Rotational Correlation Time; tr = 1/6Dr tr = 4phrh3/ 3kT Small Protein - EGF Nitroxide in H20 Large Protein – 1 MDa

  14. Electron Spin-Spin Relaxation - Accessibility Nitroxide – slow relaxation T1e’s of isolated Nitroxide spin labels are on the order of a few msec’s at physiological temperatures

  15. Electron Spin-Spin Relaxation - Accessibility Nitroxide – slow relaxation Nicklel Complexes – fast relaxation Other Transition Metals and O2

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