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Title: Orientation of the cardiac myosin regulatory light chain determined by polarized fluorescence
Author: Kampourakis, Thomas
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2012
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The regulatory light chain (RLC) of myosin is a component of the lever arm of the myosin motor in muscle thick filaments. Phosphorylation of RLC by myosin light chain kinase (MLCK) modulates the force and speed of muscle contraction, but the mechanisms of RLC‐mediated regulation in striated muscles are less well understood than those of Ca2+‐dependent regulation via troponin in the thin filament. To help elucidate the role of RLC in muscle regulation, its orientation in the native environment of cardiac muscle cells was measured. Pairs of cysteine residues were genetically introduced into the N‐ and C‐terminal lobe of the human cardiac RLC. Each pair of cysteines was crosslinked with a bifunctional‐rhodamine (BSR). The pure BSR‐RLC conjugates were exchanged into demembranated trabeculae from rat ventricle, and the orientation of the BSR fluorescence dipole determined by polarized fluorescence. The orientations of the N‐lobe were similar to those determined from chicken gizzard RLC probes exchanged into skeletal muscle fibers indicating a conserved domain orientation. The orientation of the RLC C ‐ lobe was similar in relaxation, active isometric contraction and rigor, suggesting that either the orientation of the RLC is relatively insensitive to strong binding of myosin heads to actin, or that only a small fraction of myosin heads are strongly bound to the thin filament in both active isometric contraction and rigor. Bending between the two RLC lobes occurs in ‐ situ and may have a functional significance in cardiac muscle contraction and regulation. Expressed and purified catalytic subunit of human cardiac MLCK efficiently mono ‐ phosphorylates cardiac RLC on serine 15 in a calcium/calmodulin dependent manner. Exchange of in ‐ vitro phosphorylated BSR‐RLCs into demembranated trabeculae to replace 10‐15% of native RLC showed that the orientation of phosphorylated RLC C ‐ lobe is similar to that of unphosphorylated RLCs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available