Functional characterization of skeletal F‐actin labeled on the NH2‐terminal segment of residues 1–28

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Abstract
Rabbit skeletal α‐actin was covalently labeled in the filamentous state by the fluorescent nucleophile, N‐(5‐sulfo‐1‐naphthyl)ethylenediamine (EDANS) in the presence of the carboxyl group activator 1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide (EDC). The coupling reaction was continued until the incorporation of nearly 1 mol EDANS/mol actin. After limited proteolytic digestion of the labeled protein and chromatographic identification of the EDANS‐peptides, about 80% of the attached fluorophore was found on the actin segment of residues 1–28, most probably within the N‐terminal acidic region of residues 1–7. A minor labeling site was located on the segment that consists of residues 40–113. No label was incorporated into the COOH‐terminal moiety consisting of residues 113–375. The isolated EDANS—G‐actin undergoes polymerization in the presence of salts but at a rate significantly greater than unlabeled actin. The EDANS—F‐actin could be complexed to skeletal chymotryptic myosin subfragment 1 (S‐1) and to tropomyosin. The complex formed between EDANS—F‐actin and S‐1 could not be further crosslinked by EDC but the two proteins were readily joined by glutaraldehyde as observed for native actin—S‐1, suggesting that the EDANS‐substituted carboxyl site is also involved in the EDC crosslinking of native actin to S‐1. Moreover, the EDANS labeling of F‐actin resulted in a 20‐fold increase in the Km of the actin‐activated Mg2+. ATPase of S‐1. Thus, this labeling, while it did not much affect the rigor actin—S‐1 interaction, changes the actin binding to the S‐1—nucleotide complexes significantly. The selective introduction of a variety of spectral probes, like EDANS, or other classes of fluorophores, on the N‐terminal region of actin, through the reported carbodiimide coupling reaction, would provide several different derivatives valuable for assessing the functional role of the negatively charged N‐terminus of actin during its interaction with myosin and other actin‐binding proteins.