Orientation of the essential light chain region of myosin in relaxed, active, and rigor muscle |
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Authors: | Knowles Andrea C Ferguson Roisean E Brandmeier Birgit D Sun Yin-Biao Trentham David R Irving Malcolm |
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Institution: | * Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom † Medical Research Council National Institute for Medical Research, London, United Kingdom |
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Abstract: | The orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle (β) of 120-125° with the filament axis. This is ∼30° larger than the broader distribution determined previously from RLC probes, suggesting that, relative to crystallographic structures, the LCD is bent between its ELC and RLC regions in rigor muscle. The ELC orientation distribution in relaxed muscle had two broad peaks with β ∼70° and ∼110°, which may correspond to the two head regions of each myosin molecule, in contrast with the single broad distribution of the RLC region in relaxed muscle. During isometric contraction the ELC orientation distribution peaked at β ∼105°, similar to that determined previously for the RLC region. |
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Keywords: | BR and BSR bifunctional carborhodamine and bifunctional sulforhodamine moieties respectively attached to proteins after two-site labeling with BR-I2 or BSR-I2 (bis-((N- iodoacetyl)piperazinyl) sulforhodamine-I2) reagents BSA bovine serum albumin CrP creatine phosphate DTT dithiothreitol ELC myosin essential light chain ELC-BSR mutant ELC labeled with BSR-I2 FPLC fast protein liquid chromatography GST glutathione S-transferase HPLC high performance liquid chromatography KPr potassium propionate LCD light-chain domain of myosin MgAc2 magnesium acetate ME maximum entropy PBS phosphate-buffered saline PMSF phenylmethylsulfonyl fluoride RLC myosin regulatory light chain TFP trifluoperazine PDB Protein Data Bank |
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