Subunit interactions within an expressed regulatory domain of chicken skeletal myosin. Location of the NH2 terminus of the regulatory light chain by fluorescence resonance energy transfer.

The Journal of Biological Chemistry
L D Saraswat, Susan Lowey

Abstract

The regulatory domain (RD), or neck region of the myosin head, consists of two classes of light chains that stabilize an alpha-helical segment of the heavy chain. RD from chicken skeletal muscle myosin was prepared in Escherichia coli by coexpression of a 9-kDa heavy chain fragment with the essential light chain. Recombinant regulatory light chain (RLC), wild type or mutant, was added separately to reconstitute the complex. The affinity of RD for divalent cations was determined by measuring the change in fluorescence of a pair of heavy chain tryptophans upon addition of calcium or magnesium. The complex bound divalent cations with high affinity, similar to the association constants determined for native myosin. The intrinsic fluorescence of the tryptophans could be used as a donor to measure the fluorescence resonance energy transfer distance to a single labeled cysteine engineered at position 2 on RLC. Dansylated Cys2 could also serve as a donor by preparing RLC with a second cysteine at position 79 which was labeled with an acceptor probe. These fluorescence resonance energy transfer distances (24-30 A), together with a previous measurement between Cys2 and Cys155 (Wolff-Long, V. L., Tao, T., and Lowey, S. (1995) J. Biol. Che...Continue Reading

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Apr 21, 2010·Proceedings of the National Academy of Sciences of the United States of America·David KastDavid D Thomas
May 7, 2009·Annual Review of Biophysics·David D ThomasVicci L Korman
Dec 5, 2006·American Journal of Physiology. Heart and Circulatory Physiology·Olga M HernandezDanuta Szczesna-Cordary
Aug 18, 2001·The Journal of Biological Chemistry·S S RosenfeldP H King

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