Luminescence resonance energy transfer measurements in myosin.
Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 A, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.
Burmeister Getz
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E
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Cooke
R
R
Selvin
P R
PR
D000199:Actins / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002614:Chelating Agents; D003545:Cysteine; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D049449:Luminescence; D008674:Metals, Rare Earth; D018482:Muscle, Skeletal; D018994:Myosin Light Chains / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation; D011817:Rabbits; D012235:Rhodamines; D013725:Terbium
D000199:Actins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D008674:Metals, Rare Earth; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D012235:Rhodamines; D013725:Terbium; C034996:tetramethylrhodamine iodoacetamide; D009218:Myosins; D003545:Cysteine