Bifunctional rhodamine probes of Myosin regulatory light chain orientation in relaxed skeletal muscle fibers.
The orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteine residues introduced into the RLC. Pure 1:1 BR-RLC complexes were exchanged into single muscle fibers in EDTA rigor solution for 30 min at 30 degrees C; approximately 60% of the native RLC was removed and stoichiometrically replaced by BR-RLC, and >85% of the BR-RLC was located in the sarcomeric A-bands. The second- and fourth-rank order parameters of the orientation distributions of BR dipoles linking RLC cysteine pairs 100-108, 100-113, 108-113, and 104-115 were calculated from polarized fluorescence intensities, and used to determine the smoothest RLC orientation distribution-the maximum entropy distribution-consistent with the polarized fluorescence data. Maximum entropy distributions in relaxed muscle were relatively broad. At the peak of the distribution, the "lever" axis, linking Cys707 and Lys843 of the myosin heavy chain, was at 70-80 degrees to the fiber axis, and the "hook" helix (Pro830-Lys843) was almost coplanar with the fiber and lever axes. The temperature and ionic strength of the relaxing solution had small but reproducible effects on the orientation of the RLC region.
Brack
Andrew S
AS
School of Biomedical Sciences, Guy's Campus, King's College London, London SE1 1UL, United Kingdom.
Brandmeier
Birgit D
BD
Ferguson
Roisean E
RE
Criddle
Susan
S
Dale
Robert E
RE
Irving
Malcolm
M
D000818:Animals; D002645:Chickens; D005454:Fluorescence Polarization / Q000379:methods; D008958:Models, Molecular; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000502:physiology*; D018994:Myosin Light Chains / Q000737:chemistry / Q000502:physiology*; D009218:Myosins / Q000737:chemistry / Q000502:physiology; D011817:Rabbits; D012235:Rhodamines / Q000737:chemistry*; D012518:Sarcomeres / Q000737:chemistry / Q000502:physiology
D018994:Myosin Light Chains; D012235:Rhodamines; D009218:Myosins