Interaction of myosin LYS-553 with the C-terminus and DNase I-binding loop of actin examined by fluorescence resonance energy transfer.
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 Förster distance (R(0)) was determined to be 44 and 38 A 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 A and 24.0 +/- 1.6 A, respectively. MgADP had no significant effect on the distances observed in rigor. Thus, rearrangements 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.
Yengo
C M
CM
Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, Vermont 05405-0068, USA.
Chrin
L R
LR
Berger
C L
CL
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003619:Dansyl Compounds; D003850:Deoxyribonuclease I / Q000378:metabolism; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D046911:Macromolecular Substances; D008958:Models, Molecular; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D009282:Naphthalenesulfonates; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D000244:Adenosine Diphosphate; C006421:1,5-I-AEDANS; D003850:Deoxyribonuclease I; D009218:Myosins; D008239:Lysine