PRODUCTION AND PROPERTIES OF SKELETAL MYOSIN SUBFRAGMENT-1 SELECTIVELY LABELED WITH FLUORESCEIN AT LYSINE-553 PROXIMAL TO THE STRONG ACTIN-BINDING SITE

We describe, for the first time, the reaction of skeletal myosin subfragment 1 (S-1) with the succinimido ester of 6-[fluorescein-5(and 6)-carboxamido]hexanoic acid (FHS), which takes place at pH 7.0, 20 degrees C, within a 15 min period, in the presence of 1.5-1.8-fold molar excess of reagent over protein, As a result, 0.9-1.0 mol of fluorescyl group/mol of S-1 was covalently incorporated exclusively into the 95 kDa heavy chain as monitored by spectroscopic measurements, The central 50 kDa segment included the main site of fluorescence attachment as assessed by gel electrophoresis. The extent of S-1-FHS conjugation is strongly sensitive to F-actin binding but not to the interaction of nucleotides. The formation of the rigor F-actin-S-1 complex decreased the level of S-1 labeling to 20% without any competition between actin and S-1 for FHS binding. The derivatization of S-1 did not alter the K+-ATPase activity, but it enhanced the Ca2+-ATPase and Mg2+-ATPase to 150% and 225%, respectively, whereas it lowered the actin-activated ATPase to only 75% of the original activity. A double-reciprocal plot of the ATPase rate against actin concentration indicated a 2-fold decrease of the V-max value for modified S-1, while the K-m for actin was unchanged. Cosedimentation experiments did not reveal disruption of the rigor acto-S-1 interaction by the bound fluorophore. The labeled S-1 heavy chain was isolated, and its total tryptic digest was fractionated by reverse-phase HPLC. Only two fluorescent peptides, designated P-1 and P-2, containing 15% and 85%, respectively, of the initial fluorescence were found, and after purification they were entirely sequenced. The major P-2 peptide spanned the heavy chain sequence Ala-545-Lys-561 with Lys-553 identified as the FHS-hyperreactive residue; the sequence of the minor P-1 peptide corresponded to Gly-638-Lys-641 with Lys-640 being linked to FHS. The location of Lys-553 in the S-1 primary structure is of particular interest as it is relevant to the primary stereospecific and hydrophobic actin-binding site thought to involve the helix(Gly-516-Phe-542)-loop(Pro-543-Thr-546)-helix(Asp-547-His-558) motif residing in the lower subdomain of the 50 kDa region. Lys-553 is positioned at the end of the latter helix, and the fluorescyl group bound to it may represent a valuable landmark to probe the functioning and orientational Properties of this strategic S-1 area during the acto-S-1-ATP interactions.