title: Distance measurement between the active site and cysteine-177 of the alkali one light chain of subfragment 1 from rabbit skeletal muscle.
abstract: Förster energy-transfer techniques have been applied to labeled myosin subfragment 1 from rabbit skeletal muscle to determine an intramolecular distance and whether this distance changes during magnesium-dependent ATPase activity. The alkali one light chain was labeled at Cys-177 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and then exchanged into subfragment 1. High specificity of labeling was indicated by high-performance liquid chromatography analysis of a tryptic digest of the labeled light chain. 2'(3')-O-(2,4,6-Trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was bound to the labeled protein at the ATPase active site. The efficiency of energy transfer between the probes was 0.09 when measured by both steady-state and time-resolved fluorescence. Anisotropy measurements of the bound AEDANS indicated considerable freedom of motion of the probe. The probable distance between the probes was 57 A. This distance was unchanged during triphosphatase activity. Two further sites of TNP-ADP interaction with subfragment 1 were found. The effect of these interactions on the energy-transfer measurements was reduced to a minimum by careful choice of reaction conditions.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004735:Energy Transfer; D009132:Muscles / Q000032:analysis*; D015879:Myosin Subfragments; D009218:Myosins / Q000032:analysis*; D009282:Naphthalenesulfonates / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis*; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
chemical_list: D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; C006421:1,5-I-AEDANS; D014357:Trypsin; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D003545:Cysteine