Cargando…
Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle
The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Biophysical Society
2015
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302210/ https://www.ncbi.nlm.nih.gov/pubmed/25606679 http://dx.doi.org/10.1016/j.bpj.2014.11.049 |
_version_ | 1782353756260663296 |
---|---|
author | Kampourakis, Thomas Sun, Yin-Biao Irving, Malcolm |
author_facet | Kampourakis, Thomas Sun, Yin-Biao Irving, Malcolm |
author_sort | Kampourakis, Thomas |
collection | PubMed |
description | The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1–N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1–C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface. |
format | Online Article Text |
id | pubmed-4302210 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | The Biophysical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-43022102015-07-10 Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle Kampourakis, Thomas Sun, Yin-Biao Irving, Malcolm Biophys J Molecular Machines, Motors, and Nanoscale Biophysics The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1–N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1–C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface. The Biophysical Society 2015-01-20 /pmc/articles/PMC4302210/ /pubmed/25606679 http://dx.doi.org/10.1016/j.bpj.2014.11.049 Text en © 2015 The Authors http://creativecommons.org/licenses/by/3.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Molecular Machines, Motors, and Nanoscale Biophysics Kampourakis, Thomas Sun, Yin-Biao Irving, Malcolm Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle |
title | Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle |
title_full | Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle |
title_fullStr | Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle |
title_full_unstemmed | Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle |
title_short | Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle |
title_sort | orientation of the n- and c-terminal lobes of the myosin regulatory light chain in cardiac muscle |
topic | Molecular Machines, Motors, and Nanoscale Biophysics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302210/ https://www.ncbi.nlm.nih.gov/pubmed/25606679 http://dx.doi.org/10.1016/j.bpj.2014.11.049 |
work_keys_str_mv | AT kampourakisthomas orientationofthenandcterminallobesofthemyosinregulatorylightchainincardiacmuscle AT sunyinbiao orientationofthenandcterminallobesofthemyosinregulatorylightchainincardiacmuscle AT irvingmalcolm orientationofthenandcterminallobesofthemyosinregulatorylightchainincardiacmuscle |