{"id":{"repo_id":"kings","oai_identifier":"oai:kclpure.kcl.ac.uk:studenttheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799"},"canonical_url":"https://search.dev.ndltd.org/etd/kings/oai:kclpure.kcl.ac.uk:studenttheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799","repository":{"repo_id":"kings","name":"King's College London","base_url":"https://kclpure.kcl.ac.uk/ws/oai"},"display":{"title":"Orientation of the Cardiac Myosin Regulatory Light Chain Determined by Polarized Fluorescence","abstract":"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.","abstract_html":"The regulatory light chain (RLC) of myosin is a component of the lever arm of the<br/>myosin motor in muscle thick filaments. Phosphorylation of RLC by myosin light<br/>chain kinase (MLCK) modulates the force and speed of muscle contraction, but the mechanisms of RLC‐mediated regulation in striated muscles are less well<br/>understood than those of Ca2+‐dependent regulation via troponin in the thin<br/>filament.<br/>To help elucidate the role of RLC in muscle regulation, its orientation in the native<br/>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.<br/>Each pair of cysteines was crosslinked with a bifunctional‐rhodamine (BSR). The<br/>pure BSR‐RLC conjugates were exchanged into demembranated trabeculae from rat ventricle, and the orientation of the BSR fluorescence dipole determined by<br/>polarized fluorescence.<br/>The orientations of the N‐lobe were similar to those determined from chicken<br/>gizzard RLC probes exchanged into skeletal muscle fibers indicating a conserved<br/>domain orientation. The orientation of the RLC C ‐ lobe was similar in relaxation,<br/>active isometric contraction and rigor, suggesting that either the orientation of the<br/>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<br/>active isometric contraction and rigor. Bending between the two RLC lobes occurs<br/>in ‐ situ and may have a functional significance in cardiac muscle contraction and<br/>regulation.<br/>Expressed and purified catalytic subunit of human cardiac MLCK efficiently mono ‐<br/>phosphorylates cardiac RLC on serine 15 in a calcium/calmodulin dependent<br/>manner. Exchange of in ‐ vitro phosphorylated BSR‐RLCs into demembranated<br/>trabeculae to replace 10‐15% of native RLC showed that the orientation of<br/>phosphorylated RLC C ‐ lobe is similar to that of unphosphorylated RLCs.","abstract_has_math":false,"creators":["Kampourakis, Thomas"],"institution":"King's College London","degree_name":"Doctor of Philosophy","degree_level":"Doctoral Thesis","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Irving, Malcolm","Gautel, Mathias Sebastian"],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012-10-1","date_published":"2012-10-1","updated_at":"2026-07-24T02:44:40Z","subjects":[],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["oai:kclpure.kcl.ac.uk:studenttheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799"],"render_values":[{"text":"oai:kclpure.kcl.ac.uk:studenttheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799","href":null,"code":true}]}]},"links":{"outbound_url":"https://kclpure.kcl.ac.uk/portal/en/studentTheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Irving, Malcolm","Gautel, Mathias Sebastian"]},{"key":"dc:creator","label":"Author","values":["Kampourakis, Thomas"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2012-10-1"]},{"key":"dc:date.issued","label":"Date","values":["2012-10-1"]},{"key":"dc:publisher.department","label":"Dc Publisher Department","values":["Randall Centre of Cell & Molecular Biophysics"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["King's College London"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://kclpure.kcl.ac.uk/portal/en/studentTheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral Thesis"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["oai:kclpure.kcl.ac.uk:studenttheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799","https://kclpure.kcl.ac.uk/portal/en/studentTheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://kclpure.kcl.ac.uk/portal/files/12780837/Studentthesis-Thomas_Kampourakis_2012.pdf"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:title","label":"Title","values":["Orientation of the Cardiac Myosin Regulatory Light Chain Determined by Polarized Fluorescence"]}]}],"canonical_facts":{"dc:contributor.advisor":["Irving, Malcolm","Gautel, Mathias Sebastian"],"dc:creator":["Kampourakis, Thomas"],"dc:date":["2012-10-1"],"dc:date.issued":["2012-10-1"],"dc:description.abstract":["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."],"dc:identifier":["oai:kclpure.kcl.ac.uk:studenttheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799","https://kclpure.kcl.ac.uk/portal/en/studentTheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799"],"dc:identifier.uri":["https://kclpure.kcl.ac.uk/portal/files/12780837/Studentthesis-Thomas_Kampourakis_2012.pdf"],"dc:language":["eng"],"dc:publisher.department":["Randall Centre of Cell & Molecular Biophysics"],"dc:publisher.institution":["King's College London"],"dc:relation.isreferencedby":["https://kclpure.kcl.ac.uk/portal/en/studentTheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799"],"dc:title":["Orientation of the Cardiac Myosin Regulatory Light Chain Determined by Polarized Fluorescence"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral Thesis"],"dc:type.qualificationname":["Doctor of Philosophy"]},"updated_at":"2026-07-24T02:44:40Z"}