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Distance Measurements by Continuous Wave EPR Spectroscopy to Monitor Protein Folding

  • James A. Cooke
  • Louise J. Brown
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 752)

Abstract

Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) offers a powerful method for the structural analysis of protein folds. This method can be used to test and build secondary, tertiary, and quaternary structural models as well as measure protein conformational changes in solution. Insertion of two cysteine residues into the protein backbone using molecular biology methods and the subsequent labeling of the cysteine residues with a paramagnetic spin label enables the technique of EPR to be used as a molecular spectroscopic ruler. EPR measures the dipolar interaction between pairs of paramagnetic spin labels to yield internitroxide distances from which quantitative structural information on a protein fold can then be obtained. Interspin dipolar interaction between two spin labels at less than 25  Å are measured using continuous wave (CW) EPR methods. As for any low-resolution distance methods, the positioning of the spin labels and the number of distance constraints to be measured are dependent on the structural question being asked, thus a pattern approach for using distance sets to decipher structure mapping, including protein folds and conformational changes associated with biological activity, is essential. Practical guidelines and hints for the technique of SDSL-EPR are described in this chapter, including methods for spin labeling the protein backbone, CW-EPR data collection at physiological temperatures and two semiquantitative analysis methods to extract interspin distance information from the CW-EPR spectra.

Key words

Site-directed spin labeling Electron paramagnetic resonance Protein folding Continu­ous wave EPR spectroscopy Site-directed mutagenesis Interspin distances Dipolar interaction 

Notes

Acknowledgments

This work was supported by the Australian Research Council (ARC). LB is a recipient of an ARC APD fellowship and JC is a recipient of a Macquarie University Research Areas and Centres of Excellence Award (RAACE). The authors thank Mr Michael Howell for his editorial assistance.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Chemistry and Biomolecular SciencesMacquarie UniversitySydneyAustralia

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