Fluorescence of fluorescein attached to myosin SH1 distinguishes the rigor state from the actin-myosin-nucleotide state.
It has been found that the fluorescence intensity of 5-(iodoacetamido)fluorescein (5-IAF) attached to the SH1 of myosin subfragment 1 (S-1) increases 3-fold on formation of the rigor complex. On adding Mg2+-ADP, light scattering indicates no dissociation, but the fluorescence increment disappears. Thus, this fluorescence signal can distinguish the rigor state from other states, especially from ternary complexes such as actin-myosin-nucleotide. We demonstrate that by using this signal we can measure spectroscopically several kinetic parameters of acto-S-1-nucleotide interaction: In the presence of 20 mM KCl, 2 mM MgSO4, and 10 mM TES (pH 7.5) at 22 degrees C, Mg2+-ADP binds to acto-S-1(5)* (S-1(5) denotes 5-IAF-labeled S-1) with a Ka = 2 X 10(6) M-1, and Mg2+-PPi binds to acto-S-1(5)* with two apparent affinities, Ka = 8 X 10(4) M-1 and 1.4 X 10(3) M-1; the association rates of Mg2+-ADP and Mg2+-ATP for acto-S-1(5)* are 10(7) s-1 M-1 and 4 X 10(6) s-1 M-1, respectively, and the dissociation rate of Mg2+-ADP for acto-S-1(5)* is 5 s-1. In contrast to the fluorescence intensity of the dye, the lifetime and the absorbance are essentially unaffected by complex formation with F-actin or nucleotides. Therefore, we conclude that there must be a static quencher such as Trp, Tyr, or Met in the neighborhood of the attached dye and that the contact between dye and quencher is modulated by actin-induced or nucleotide-induced conformational changes in S-1.
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D005452:Fluoresceins; D007700:Kinetics; D008274:Magnesium; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D005452:Fluoresceins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; C015296:5-iodoacetamidofluorescein; D009218:Myosins; D008274:Magnesium