TY - JOUR
T1 - Response of rigor cross-bridges to stretch detected by fluorescence lifetime imaging microscopy of myosin essential light chain in skeletal muscle fibers
AU - Ushakov, Dmitry S.
AU - Caorsi, Valentina
AU - Ibanez-Garcia, Delisa
AU - Manning, Hugh B.
AU - Konitsiotis, Antonios D.
AU - West, Timothy G.
AU - Dunsby, Christopher
AU - French, Paul M.
AU - Ferenczi, Michael A.
PY - 2011/1/7
Y1 - 2011/1/7
N2 - We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)-ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.
AB - We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)-ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.
UR - http://www.scopus.com/inward/record.url?scp=78650941120&partnerID=8YFLogxK
U2 - 10.1074/jbc.M110.149526
DO - 10.1074/jbc.M110.149526
M3 - Article
C2 - 21056977
AN - SCOPUS:78650941120
SN - 0021-9258
VL - 286
SP - 842
EP - 850
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -