Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres.
Changes in the orientation of the myosin regulatory light chain (RLC) in single muscle fibres were measured using polarised fluorescence from acetamidotetramethylrhodamine (ATR). Mutants of chicken skeletal RLC containing single cysteine residues at positions 2, 73, 94, 126 and 155 were labelled with either the 5 or 6-isomer of iodo-ATR, giving ten different probes. The labelled RLCs were exchanged into demembranated fibres from rabbit psoas muscle without significant effect on active force generation. Fluorescence polarisation measurements showed that nine out of the ten probe dipoles were more perpendicular to the fibre axis in the absence of ATP (in rigor) than in either relaxation or active contraction. The orientational distribution of the RLC region of the myosin head in active contraction is closer to the relaxed than to the rigor orientation, and is not equivalent to a linear combination of the relaxed and rigor orientations. Rapid length steps were applied to the fibres to synchronise the motions of myosin heads attached to actin. In active contraction the fluorescence polarisation changed both during the step, indicating elastic distortion of the RLC region of the myosin head, and during the subsequent rapid force recovery that is thought to signal the working stroke. The peak change in fluorescence polarisation produced by an active release of 5 nm per half sarcomere indicates an axial tilt of less than 5 degrees for all ten probes, if all the myosin heads in the fibre respond to the length step. This tilting was towards the rigor orientation for all ten probes, and could be explained by 14% of the heads moving to the rigor orientation. An active stretch tilted the heads away from the rigor conformation by a similar extent.
Sabido-David
C
C
Randall Institute, King's College London, UK.
Hopkins
S C
SC
Saraswat
L D
LD
Lowey
S
S
Goldman
Y E
YE
Irving
M
M
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003545:Cysteine / Q000737:chemistry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D012235:Rhodamines
D005456:Fluorescent Dyes; D018994:Myosin Light Chains; D012235:Rhodamines; C059222:acetamidotetramethylrhodamine; D003545:Cysteine