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. 1998 Jun;74(6):3093–3110. doi: 10.1016/S0006-3495(98)78016-6

Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.

S C Hopkins 1, C Sabido-David 1, J E Corrie 1, M Irving 1, Y E Goldman 1
PMCID: PMC1299650  PMID: 9635763

Abstract

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.

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Selected References

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  1. Allen T. S., Ling N., Irving M., Goldman Y. E. Orientation changes in myosin regulatory light chains following photorelease of ATP in skinned muscle fibers. Biophys J. 1996 Apr;70(4):1847–1862. doi: 10.1016/S0006-3495(96)79750-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barabás K., Keszthelyi L. Temperature dependence of ATP release from "caged" ATP. Acta Biochim Biophys Acad Sci Hung. 1984;19(3-4):305–309. [PubMed] [Google Scholar]
  3. Berger C. L., Craik J. S., Trentham D. R., Corrie J. E., Goldman Y. E. Fluorescence polarization of skeletal muscle fibers labeled with rhodamine isomers on the myosin heavy chain. Biophys J. 1996 Dec;71(6):3330–3343. doi: 10.1016/S0006-3495(96)79526-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biophysical Society 41st annual meeting. New Orleans, Louisiana, 2-6 March 1997. Abstracts. Biophys J. 1997 Feb;72(2 Pt 2):A1–476. [PMC free article] [PubMed] [Google Scholar]
  5. Borejdo J., Putnam S. Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation. Biochim Biophys Acta. 1977 Mar 11;459(3):578–595. doi: 10.1016/0005-2728(77)90056-1. [DOI] [PubMed] [Google Scholar]
  6. Brenner B. Mechanical and structural approaches to correlation of cross-bridge action in muscle with actomyosin ATPase in solution. Annu Rev Physiol. 1987;49:655–672. doi: 10.1146/annurev.ph.49.030187.003255. [DOI] [PubMed] [Google Scholar]
  7. Brenner B., Schoenberg M., Chalovich J. M., Greene L. E., Eisenberg E. Evidence for cross-bridge attachment in relaxed muscle at low ionic strength. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7288–7291. doi: 10.1073/pnas.79.23.7288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brenner B., Yu L. C., Podolsky R. J. X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths. Biophys J. 1984 Sep;46(3):299–306. doi: 10.1016/S0006-3495(84)84026-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burghardt T. P., Garamszegi S. P., Ajtai K. Probes bound to myosin Cys-707 rotate during length transients in contraction. Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9631–9636. doi: 10.1073/pnas.94.18.9631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. CHEN R. F., BOWMAN R. L. FLUORESCENCE POLARIZATION: MEASUREMENT WITH ULTRAVIOLET-POLARIZING FILTERS IN A SPECTROPHOTOFLUOROMETER. Science. 1965 Feb 12;147(3659):729–732. doi: 10.1126/science.147.3659.729. [DOI] [PubMed] [Google Scholar]
  11. Cecchi G., Colomo F., Lombardi V. A loudspeaker servo system for determination of mechanical characteristics of isolated muscle fibres. Boll Soc Ital Biol Sper. 1976 May 30;52(10):733–736. [PubMed] [Google Scholar]
  12. Chase P. B., Kushmerick M. J. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys J. 1988 Jun;53(6):935–946. doi: 10.1016/S0006-3495(88)83174-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cooke R., Crowder M. S., Thomas D. D. Orientation of spin labels attached to cross-bridges in contracting muscle fibres. Nature. 1982 Dec 23;300(5894):776–778. doi: 10.1038/300776a0. [DOI] [PubMed] [Google Scholar]
  14. Cooke R., Crowder M. S., Wendt C. H., Barnett V. A., Thomas D. D. Muscle cross-bridges: do they rotate? Adv Exp Med Biol. 1984;170:413–427. doi: 10.1007/978-1-4684-4703-3_37. [DOI] [PubMed] [Google Scholar]
  15. Cooke R. The mechanism of muscle contraction. CRC Crit Rev Biochem. 1986;21(1):53–118. doi: 10.3109/10409238609113609. [DOI] [PubMed] [Google Scholar]
  16. Finer J. T., Simmons R. M., Spudich J. A. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature. 1994 Mar 10;368(6467):113–119. doi: 10.1038/368113a0. [DOI] [PubMed] [Google Scholar]
  17. Goldman Y. E., Hibberd M. G., Trentham D. R. Initiation of active contraction by photogeneration of adenosine-5'-triphosphate in rabbit psoas muscle fibres. J Physiol. 1984 Sep;354:605–624. doi: 10.1113/jphysiol.1984.sp015395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldman Y. E., Hibberd M. G., Trentham D. R. Relaxation of rabbit psoas muscle fibres from rigor by photochemical generation of adenosine-5'-triphosphate. J Physiol. 1984 Sep;354:577–604. doi: 10.1113/jphysiol.1984.sp015394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goldman Y. E. Measurement of sarcomere shortening in skinned fibers from frog muscle by white light diffraction. Biophys J. 1987 Jul;52(1):57–68. doi: 10.1016/S0006-3495(87)83188-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldman Y. E., Simmons R. M. Control of sarcomere length in skinned muscle fibres of Rana temporaria during mechanical transients. J Physiol. 1984 May;350:497–518. doi: 10.1113/jphysiol.1984.sp015215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gollub J., Cremo C. R., Cooke R. ADP release produces a rotation of the neck region of smooth myosin but not skeletal myosin. Nat Struct Biol. 1996 Sep;3(9):796–802. doi: 10.1038/nsb0996-796. [DOI] [PubMed] [Google Scholar]
  22. Huxley A. F., Simmons R. M. Proposed mechanism of force generation in striated muscle. Nature. 1971 Oct 22;233(5321):533–538. doi: 10.1038/233533a0. [DOI] [PubMed] [Google Scholar]
  23. Irving M., Lombardi V., Piazzesi G., Ferenczi M. A. Myosin head movements are synchronous with the elementary force-generating process in muscle. Nature. 1992 May 14;357(6374):156–158. doi: 10.1038/357156a0. [DOI] [PubMed] [Google Scholar]
  24. Irving M., St Claire Allen T., Sabido-David C., Craik J. S., Brandmeier B., Kendrick-Jones J., Corrie J. E., Trentham D. R., Goldman Y. E. Tilting of the light-chain region of myosin during step length changes and active force generation in skeletal muscle. Nature. 1995 Jun 22;375(6533):688–691. doi: 10.1038/375688a0. [DOI] [PubMed] [Google Scholar]
  25. Irving M. Steady-state polarization from cylindrically symmetric fluorophores undergoing rapid restricted motion. Biophys J. 1996 Apr;70(4):1830–1835. doi: 10.1016/S0006-3495(96)79748-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ling N., Shrimpton C., Sleep J., Kendrick-Jones J., Irving M. Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle. Biophys J. 1996 Apr;70(4):1836–1846. doi: 10.1016/S0006-3495(96)79749-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lombardi V., Piazzesi G., Ferenczi M. A., Thirlwell H., Dobbie I., Irving M. Elastic distortion of myosin heads and repriming of the working stroke in muscle. Nature. 1995 Apr 6;374(6522):553–555. doi: 10.1038/374553a0. [DOI] [PubMed] [Google Scholar]
  28. Lombardi V., Piazzesi G. The contractile response during steady lengthening of stimulated frog muscle fibres. J Physiol. 1990 Dec;431:141–171. doi: 10.1113/jphysiol.1990.sp018324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lowey S., Waller G. S., Trybus K. M. Skeletal muscle myosin light chains are essential for physiological speeds of shortening. Nature. 1993 Sep 30;365(6445):454–456. doi: 10.1038/365454a0. [DOI] [PubMed] [Google Scholar]
  30. Martyn D. A., Chase P. B. Faster force transient kinetics at submaximal Ca2+ activation of skinned psoas fibers from rabbit. Biophys J. 1995 Jan;68(1):235–242. doi: 10.1016/S0006-3495(95)80179-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Molloy J. E., Burns J. E., Kendrick-Jones J., Tregear R. T., White D. C. Movement and force produced by a single myosin head. Nature. 1995 Nov 9;378(6553):209–212. doi: 10.1038/378209a0. [DOI] [PubMed] [Google Scholar]
  32. Ostap E. M., Barnett V. A., Thomas D. D. Resolution of three structural states of spin-labeled myosin in contracting muscle. Biophys J. 1995 Jul;69(1):177–188. doi: 10.1016/S0006-3495(95)79888-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rayment I., Holden H. M., Whittaker M., Yohn C. B., Lorenz M., Holmes K. C., Milligan R. A. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 1993 Jul 2;261(5117):58–65. doi: 10.1126/science.8316858. [DOI] [PubMed] [Google Scholar]
  34. Rayment I., Rypniewski W. R., Schmidt-Bäse K., Smith R., Tomchick D. R., Benning M. M., Winkelmann D. A., Wesenberg G., Holden H. M. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science. 1993 Jul 2;261(5117):50–58. doi: 10.1126/science.8316857. [DOI] [PubMed] [Google Scholar]
  35. Roopnarine O., Thomas D. D. Orientational dynamics of indane dione spin-labeled myosin heads in relaxed and contracting skeletal muscle fibers. Biophys J. 1995 Apr;68(4):1461–1471. doi: 10.1016/S0006-3495(95)80319-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sabido-David C., Brandmeier B., Craik J. S., Corrie J. E., Trentham D. R., Irving M. Steady-state fluorescence polarization studies of the orientation of myosin regulatory light chains in single skeletal muscle fibers using pure isomers of iodoacetamidotetramethylrhodamine. Biophys J. 1998 Jun;74(6):3083–3092. doi: 10.1016/S0006-3495(98)78015-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sabido-David C., Hopkins S. C., Saraswat L. D., Lowey S., Goldman Y. E., Irving M. Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres. J Mol Biol. 1998 Jun 5;279(2):387–402. doi: 10.1006/jmbi.1998.1771. [DOI] [PubMed] [Google Scholar]
  38. Tanner J. W., Thomas D. D., Goldman Y. E. Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres. J Mol Biol. 1992 Jan 5;223(1):185–203. doi: 10.1016/0022-2836(92)90725-y. [DOI] [PubMed] [Google Scholar]
  39. Tregear R. T., Mendelson R. A. Polarization from a helix of fluorophores and its relation to that obtained from muscle. Biophys J. 2009 Jan 1;15(5):455–467. doi: 10.1016/S0006-3495(75)85830-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Uyeda T. Q., Abramson P. D., Spudich J. A. The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4459–4464. doi: 10.1073/pnas.93.9.4459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. VanBuren P., Waller G. S., Harris D. E., Trybus K. M., Warshaw D. M., Lowey S. The essential light chain is required for full force production by skeletal muscle myosin. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12403–12407. doi: 10.1073/pnas.91.26.12403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Walker J. W., Reid G. P., Trentham D. R. Synthesis and properties of caged nucleotides. Methods Enzymol. 1989;172:288–301. doi: 10.1016/s0076-6879(89)72019-x. [DOI] [PubMed] [Google Scholar]
  43. Whittaker M., Wilson-Kubalek E. M., Smith J. E., Faust L., Milligan R. A., Sweeney H. L. A 35-A movement of smooth muscle myosin on ADP release. Nature. 1995 Dec 14;378(6558):748–751. doi: 10.1038/378748a0. [DOI] [PubMed] [Google Scholar]
  44. Xie X., Harrison D. H., Schlichting I., Sweet R. M., Kalabokis V. N., Szent-Györgyi A. G., Cohen C. Structure of the regulatory domain of scallop myosin at 2.8 A resolution. Nature. 1994 Mar 24;368(6469):306–312. doi: 10.1038/368306a0. [DOI] [PubMed] [Google Scholar]

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