NDLI: Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin

Content Provider	PubMed Central
Author	Nelson, Wendy D. Blakely, Sarah E. Nesmelov, Yuri E. Thomas, David D.
Copyright Year	2005
Abstract	We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head–head interactions, activating myosin in smooth muscle.
Related Links	http://dx.doi.org/10.1073/pnas.0401664102
Ending Page	4005
Page Count	6
Starting Page	4000
File Format	PDF
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	11
Volume Number	102
Language	English
Publisher	National Academy of Sciences
Publisher Date	2005-03-15
Access Restriction	Open
Rights Holder	National Academy of Sciences
Subject Keyword	General Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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