Bifunctional Spin Labeling of Muscle Proteins: Accurate Rotational Dynamics, Orientation, and Distance by EPR

Andrew R. Thompson, Benjamin P. Binder, Jesse E. McCaffrey, Bengt Svensson, David D. Thomas

Research output: Chapter in Book/Report/Conference proceedingChapter

10 Scopus citations


While EPR allows for the characterization of protein structure and function due to its exquisite sensitivity to spin label dynamics, orientation, and distance, these measurements are often limited in sensitivity due to the use of labels that are attached via flexible monofunctional bonds, incurring additional disorder and nanosecond dynamics. In this chapter, we present methods for using a bifunctional spin label (BSL) to measure muscle protein structure and dynamics. We demonstrate that bifunctional attachment eliminates nanosecond internal rotation of the spin label, thereby allowing the accurate measurement of protein backbone rotational dynamics, including microsecond-to-millisecond motions by saturation transfer EPR. BSL also allows for accurate determination of helix orientation and disorder in mechanically and magnetically aligned systems, due to the label's stereospecific attachment. Similarly, labeling with a pair of BSL greatly enhances the resolution and accuracy of distance measurements measured by double electron-electron resonance (DEER). Finally, when BSL is applied to a protein with high helical content in an assembly with high orientational order (e.g., muscle fiber or membrane), two-probe DEER experiments can be combined with single-probe EPR experiments on an oriented sample in a process we call BEER, which has the potential for ab initio high-resolution structure determination.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Number of pages23
StatePublished - 2015

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Publisher Copyright:
© 2015 Elsevier Inc.


  • BSL
  • Bicelles
  • DEER
  • Electron paramagnetic resonance
  • Myosin
  • Phospholamban
  • Protein dynamics
  • Protein orientation
  • Protein structure
  • Saturation transfer EPR


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