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Time Domain EPR: Membrane-binding Proteins

Time Domain EPR: Membrane-binding Proteins. Graduate Students: Tamara Okonogi Robert Nielsen. Post Docs: Andy Ball Ying Lin Stephane Canaan Kepeng Che. Faculty: Michael Gelb Kate Pratt.

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Time Domain EPR: Membrane-binding Proteins

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  1. Time Domain EPR: Membrane-binding Proteins Graduate Students: Tamara Okonogi Robert Nielsen Post Docs: Andy Ball Ying Lin Stephane Canaan Kepeng Che Faculty: Michael Gelb Kate Pratt Using R1 from EPR as a probe of the Structure-function and the Dynamics-function relation in biology Supported by NSF and NIH

  2. Time Domain EPR: Outline • Time Domain: Saturation Recovery and Pulsed Electron Double Resonance Methods • Comparison with CW methods • Spectrometer, experiment, data • Theory of relaxation Rates • Application to the Spin Relaxant Method • Using site directed mutagenesis • Orienting a Membrane-binding Protein • Determine an Oxygen gradient in a membrane

  3. CW Power Saturation The method to obtain spin-lattice relaxation rates using CW methods. Plot the Peak to Peak height as a function of microwave power (or really amplitude).

  4. Details of CW Power Saturation Peak-to-Peak height A product of spin-spin and spin-lattice relaxation rates.

  5. CW Field (Gauss)

  6. TD ESR Spectrometer

  7. Pulsed Bridge

  8. Free Induction Decay Measures spin-spin dephasing

  9. Pulsed Saturation Recovery Measures relaxation to equilibrium

  10. Pulsed Electron-Electron Double Resonance

  11. pSR; the effect of a relaxant

  12. Collision with Oxygen

  13. Redfield Theory (or BWRT) Relaxation rate theory began with Bloch and Wangness, and was amplified by Alfred Redfield to be a complete theory for the effects of dynamics and stochastic processes on spins in condensed matter. Relaxation theory is often called BWRT (Bloch Wangness and Redfield Theory). BWRT Predicts Spin-Lattice (R1) and Spin-Spin (R2) relaxation rates. Relaxation Rates are related to Relaxation times:

  14. What Redfield Theory (BWRT) Uses • There is a system Hamiltonian • BWRT requires a bilinear operator, which couples the spin system (S) to the lattice (or bath), F • The Hamiltonian is: • F is the fluctuating variable that causes the spins to have a fluctuating environment • The fluctuation of the lattice, coupled to the spin system, then causes the spins to relax or dissipate the absorbed (microwave or r.f.) energy, non-radiatively.

  15. Nielsen, R. D. and Robinson, B. H. "A Novel Relaxation Equation of Motion". J.Physical Chemistry 2004; 108: 1589-1600. A problem: R2 diverges The coupling Hamiltonian (e.n.d.) is: The orientation variable, , is a stochastic function of time. The correlation function (at high temperature) is: This shows the statistical origin of the rotational correlation time. Exponential decay of the correlation function with time is typical of such functions.

  16. CW Spectra CTPO

  17. D.A.Haas, C. Mailer, and B.H. Robinson, Biophysical J. (1993) 64, 594 R2 from BWRT, diverging R2 rates from Kubo Theory R2 rates from modified BWRT Relaxation Rates (MHz)

  18. R1 does not diverge • R1 for the electron and R1 for the nitrogen nucleus in a nitroxide spin label as a function of rotational correlation times can be computed from BWRT. • If R2 diverges for correlation times longer than a few nanoseconds how can we rely on the theory to give us R1 values out to milliseconds and beyond? • The Problem: Why does the theory fail for R2 rates but not for R1 rates? • It is important to understand why R1 works and to understand why R2 fails.

  19. Nitroxide Nitrogen Spin Lattice Relaxation Rates Electron spin-lattice relaxation rates: With O2 and Without O2 Relaxation Rates (sec-1) Correlation Time (sec) B.H. Robinson, D. A. Haas and C. Mailer (1994). Science263(5146): 490-3.

  20. Mechanisms of R1 • Sum the rates from statistically independent processes • Spin-Rotation, rate goes as • Electron-Nuclear Dipolar Coupling • Electron rate peaks at the spectrometer frequency • Nuclear rate Peaks at coupling • Oxygen relaxation (used later) • Empirical “Spin-Diffusion” process • Just a catch-all effect, goes at • Partially due to spins local to the nitroxide

  21. The ideal form of the solution is: The actual form of the solution is a bit more complicated: The two rates are: The slower rate dominates. Solution (Signal) Black: two rate solution Blue: rate Green: BWRT rate Time (sec)

  22. Dominant Rate in all limits In the slow motion limit: In the fast motion limit: BWRT gives only the fast motion limit, which predicts that the rate goes to infinity as the correlation time goes to infinity. The new theory avoids this and correctly predicts coherent oscillations of (at frequency ) as the interaction becomes coherent, in the no motion limit. The rates in the two limits may be “combined” into a rate that does cover both motional regimes (for both R1 and R2): New Term in both R2 and R1 rates

  23. Spin Labeled-Fatty Acids in DOPC Spin-Lattice Relaxation rates for varied Doxyl-Steric Acids in DOPC. Data from Jin and Hyde • Different spectrometer frequencies (from 2 to 35 GHz) with the best possible single effective correlation time. • A poor fit. The frequency dependence of simple isotropic rotational motion is incomplete. SL at 5 position SL at 12 position SL at 16 position

  24. Same Data Different Model • Improving the model to include anisotropic dynamics. • For simplicity the anisotropy ratio was kept constant. • Improved agreement indicates the need to improve the model, and the frequency dependence of the relaxation rates can rule-out some incorrect models.

  25. Relaxation rates from 60 different experiments Correlation among all the data and the model. Model has 1 adjustable parameter (the mean rotational correlation time) for each sample at all 5 different frequencies and two different isotopic forms.

  26. Membrane Binding Proteins Bee venom phospholipase Oriented on a membrane surface by Site Directed Mutagenesis EPR spin relaxant method Lin, Y., Nielsen, R., Murray, D., Hubbell, W. L., Mailer, C., Robinson, B. H. and Gelb, M. H. Science 1998; 279 (5358): 1925-9

  27. Labeling a protein (PLA2) with a Spin Probe Use site directed mutagenesis techniques to prepare proteins with a single properly placed cytsteine. General Reaction for adding relaxants The protein should contain only one cysteine for labeling. Protein labeled at only one site at a time per experiment.

  28. Relaxant Method: Nitroxide Spectra depend on concentration of relaxants Rates are increased by the same amount due to additional relaxing agents (relaxants). Spin-Lattice: T1-1 or R1 Spin-Spin: T2-1 or R2

  29. Human (HGIIA) Secretory Phospholipase sPLA2 A highly charged (+20 residues) lipase, 14kDa protein And a highly charged (-70 mV) membrane All exposure data was determined by SR and pELDOR directly measuring spin-lattice relaxation rates.

  30. CW Spectra of hGIIA on Micelles CTPO hGIIA

  31. CW Spectrum of site N70C with CROX

  32. Probing the hGIIA protein surface potential using CW and TD EPR

  33. Spin Lattice Relaxation Rates for sl-sPLA2 rates from pSR and pELDOR for CTPO solvent accessibility

  34. Power Saturation Curves site S120C

  35. O2 Relaxant: hGIIA on LUV TD CW

  36. Compare O2 Relaxant Effects from TD-SR and CW

  37. Summary of Vesicle data • Large protein surface charge determined by CW and TD data • Complete protection from Crox for all EPR data • Oxygen effect reduced relative to solution • Light scattering occurs

  38. Aggregation model ~50 enzymes (36 Angstrom diameter) LUV of DOPM (100 nm diameter)

  39. TD data, Vesicles vs. Mixed Micelles Vesicle (DTPM) Mixed Micelles

  40. hGIIA-sPLA2 on mixed micelles Crox NiEDDA

  41. sPLA2 on Membrane View from membrane Yellow: Hydrophobic Residues Blue: Charged (pos) residues Orientation perpendicular to that predicted by M. Jain. Anchored by hydrophobic residues. Charges not essential

  42. sPLA2 Conclusions sPLA2 causes the vesicles to aggregate. Explains much other data and misconceptions about the kinetics and processive nature of sPLA2 action. sPLA2 was oriented on micelles (instead) using spin-lattice relaxation rates alone. Orientation different from that of another model. Hydrophobic residues are the main points of contact. Charges provide a general, non-specific attraction. Substrate binding site identified by orientation on the mixed micelles

  43. The WALP Proteins WALP proteins are single alpha helical membrane-spanning proteins. The sequence is 23 residues long: HCO-NH-G-WW-L-(AL)8-WW-A-CO-NH2 Leucine and Alanine are both hydrophobic. In a membrane this forms a single turn alpha helix. The membrane, di-oleic (DO) PC, is about 28-30 Ang thick. The two outer Tryptophans (W) are about 30 Angs apart. The membrane will stretch (or shrink) to accommodate the protein. Demmers et al: J. Biol. Chem., 276, 34501-34508, 2001

  44. WALP23 The sequence is 23 residues long: HCO-NH-G-WW-L-(AL)8-WW-A-CO-NH2 Subcyznski et al. Biochemistry, 2003, 42, 3939

  45. WALP23-sl CW spectra CW EPR spectra of spin labeled WALP23 at various positions.

  46. Oxygen Transport Parameter The Oxygen transport parameter is the change in the spin-lattice relaxation rate due to oxygen collision-relaxation , where Depends on transport properties (e.g. Diffusion) of Oxygen in the local environment of the spin label

  47. Typical WALP/DOPC Saturation Recovery EPR With Oxygen CW Without Oxygen

  48. Walp23 in DOPM: Oxygen Transport Parameter Estimated from the CW line width From SR

  49. Walp23 in DOPC: Oxygen Transport Parameter From SR Estimated from the CW line width

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