Atomistic Models from Orientation and Distance Constraints Using EPR of a Bifunctional Spin Label

Benjamin P. Binder, Andrew R. Thompson, David D. Thomas

Research output: Contribution to journalArticlepeer-review

Abstract

We have used high-resolution orientation and distance measurements derived from electron paramagnetic resonance of a bifunctional spin label (BSL) to build and refine atomistic models of protein structure. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin's catalytic domain while myosin is in complex with actin. Constraints for orientation of individual helices were obtained in a previous study from continuous-wave electron paramagnetic resonance of myosin labeled at specific sites with BSLs in oriented muscle fibers. In this study, new distance constraints were derived from double electron-electron resonance on myosin constructs labeled with a BSL specifically at two sites. Using these complementary constraints together, we thoroughly characterize the BSL's rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. We also leverage these measurements to derive a novel, to our knowledge, structural model for myosin-II in complex with actin and MgADP and compare our model to other recent actomyosin structures. The described approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di-Cys mutation is feasible.

Original languageEnglish (US)
Pages (from-to)319-330
Number of pages12
JournalBiophysical journal
Volume117
Issue number2
DOIs
StatePublished - Jul 23 2019

Bibliographical note

Funding Information:
EPR experiments were performed at the Biophysical Technology Center, University of Minnesota. This study was supported by National Institutes of Health Grants AR32961 and AG26160 (to D.D.T.). B.P.B. was supported by National Institutes of Health Grants T32 AR007612 and K12 GM119955 .

Publisher Copyright:
© 2019

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