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CW EPR and DEER Methods to Determine BCL-2 Family Protein Structure and Interactions: Application of Site-Directed Spin Labeling to BAK Apoptotic Pores

  • Tirtha Mandal
  • Eric J. Hustedt
  • Likai Song
  • Kyoung Joon Oh
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

The continuous wave (CW) and pulse electron paramagnetic resonance (EPR) methods enable the measurement of distances between spin-labeled residues in biopolymers including proteins, providing structural information. Here we describe the CW EPR deconvolution/convolution method and the four-pulse double electron–electron resonance (DEER) approach for distance determination, which were applied to elucidate the organization of the BAK apoptotic pores formed in the lipid bilayers.

Key words

Apoptotic pore Bcl-2 BAK BAX Convolution CW EPR DEER Deconvolution Distance measurement Site-directed spin labeling (SDSL) Tikhonov regularization DeerAnalysis DEFit FitGUI 

Notes

Acknowledgement

We thank Dr. Candice Klug at the National Biomedical EPR Center, Milwaukee, Wisconsin for the technical support in Q-band experiments. We also thank Drs. Gunnar Jeschke and Christian Altenbach for helpful discussions and Mr. Carter Grieve for his proofreading the manuscript. This work was supported by NIH grant R01GM097508, EPR Center of the Rosalind Franklin University of Medicine and Science, and the RFUMS Start-up fund to. K.J.O.; NIH grant P01 GM080513 to E.J.H.; NIH grants RR022422 (DEER instrumentation grant), OD011937 (DEER instrumentation grant), and EB001980 (National Biomedical EPR Center grant) to C.K; and NSF Cooperative Agreement No. DMR-1644779 to L.S.

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Authors and Affiliations

  • Tirtha Mandal
    • 2
    • 1
  • Eric J. Hustedt
    • 3
  • Likai Song
    • 4
  • Kyoung Joon Oh
    • 1
  1. 1.Department of Biochemistry & Molecular Biology, The Chicago Medical SchoolRosalind Franklin University of Medicine and ScienceNorth ChicagoUSA
  2. 2.Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA
  3. 3.Department of Molecular Physiology and BiophysicsVanderbilt University School of MedicineNashvilleUSA
  4. 4.National High Magnetic Field LaboratoryFlorida State UniversityTallahasseeUSA

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