Interaction of myosin LYS-553 with the C-terminus and DNase I-binding loop of actin examined by fluorescence resonance energy transfer

Christopher M. Yengo, Lynn R. Chrin, Christopher L. Berger

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Abstract

Fluorescence resonance energy transfer (FRET) experiments were carried out in the absence of nucleotide (rigor) or in the presence of MgADP between fluorescent donor probes (IAEDANS (5((((2-iodoacetyl)amino)ethyl)amino)-naphthalene-1-sulfonic acid) at Cys-374 or DANSYL (5-dimethylamino naphthalene-1-(N-(5-aminopentyl))sulfonamide) at Gln-41 of actin and acceptor molecules (FHS (6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester) at Lys-553 of skeletal muscle myosin subfragment 1. The critical Forster distance (R0) was determined to be 44 and 38 Å for the IAEDANS-FHS and DANSYL-FHS donor-acceptor pairs, respectively. The efficiency of energy transfer between the acceptor molecules at Lys-553 of myosin and donor probes at Cys-374 or Gln-41 of actin was calculated to be 0.78 ± 0.01 or 0.94 ± 0.01, respectively, corresponding to distances of 35.6 ± 0.4 Å, and 24.0 ± 1.6 Å, respectively. MgADP had no significant effect on the distances observed in rigor. Thus, rearrange. ments in the acto-myosin interface are likely to occur elsewhere than in the lower 50-kDa subdomain of myosin as its affinity for actin is weakened by MgADP binding. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)187-196
Number of pages10
JournalJournal of Structural Biology
Volume131
Issue number3
DOIs
StatePublished - Jan 1 2000

All Science Journal Classification (ASJC) codes

  • Structural Biology

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