Fluorescence techniques will be used to probe the topology of smooth muscle myosin. The major goal of these experiments is to answer questions about the molecular mechanism of regulation of smooth muscle activity. Fluorescent and/or chromophoric compounds will be used to specifically label the myosin active site, various positions within the heavy chain, the two different kinds of light chains, and a specific site on the rod portion of the molecule. A novel new method of attaching probes specifically to serines or threonines that are phosphorylated by kinases is presented. This labeling method has potential to be generally useful to study the structural aspects of phosphorylated sites in other systems specifically phosphorylated by kinases. Fluorescence energy transfer techniques will be used as a spectroscopic ruler to probe conformational transitions which may be important to the mechanism of smooth muscle myosin regulation by light chain phosphorylation. The effects of phosphorylation of the regulatory light chains by either myosin light chain kinase or protein kinase C upon these distances will be examined. Using these approaches, the hypothesis that the head-tail junction of the myosin molecule plays a key role in the mechanism of regulation of smooth muscle myosin will be tested. This information will be useful to determine how the phosphorylation dependent conformational changes observed in vitro relate to physiologically important structural changes within myosin filaments in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AR040917-05
Application #
2080338
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1990-09-01
Project End
1996-03-31
Budget Start
1994-09-01
Budget End
1996-03-31
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Washington State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Jackson Jr, Del R; Webb, Milad; Stewart, Travis J et al. (2014) Sucrose increases the activation energy barrier for actin-myosin strong binding. Arch Biochem Biophys 552-553:74-82