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Abstract
The NH2- and COOH-terminal regions of the regulatory light chain (LC2) have been mapped to the head/rod junction by immunoelectron microscopy, using monoclonal and anti-fluorescyl antibodies as probes. In order to map the entire length of the light chain, we have engineered and purified mutants that contain a single cysteine residue at positions 2, 73, 94, 126, or 155. The single cysteine residues were labeled with either 5-iodoacetamido fluorescein or N-ethylmaleimide-biotin. We observed that the reactivity of Cys126 is far greater than that of Cys155, confirming that cysteine 126 is the fast-reacting thiol in wild-type light chain. The labeled light chains were exchanged into myosin stripped of its native LC2 by immunoaffinity chromatography, and the reconstituted myosin was reacted with anti-fluorescein antibody or avidin prior to rotary shadowing for electron microscopy. The position of the antibody or avidin was found to be near the head/rod junction for all mutants. These mapping studies, together with our finding that cysteines widely separated in the primary sequence can form multiple disulfide bonds (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 226, 19777-19785), support a model for LC2 as a flexible, globular molecule localized mainly in the vicinity of the head/rod junction of myosin.
Highlights
From the $Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Walthum, Massachusetts 02254 and the $Department of Chemistry, Wheaton College, Norton, Massachusetts 02766
The labeled light chains were exchanged into myosin stripped of its native LC2 by immunoaffinity chromatography, and the reconstituted myosin was reacted with anti-fluorescein antibody or avidin prior to rotary shadowing for electron microscopy
These resultsindicate that thebulk of the regulatory light chain is confined to this region of sition of the antibody or avidin was found to be near myosin, and does notextendto the ATP or actin-binding the head/rod junction for all mutants
Summary
Expression and Characterization of LC2 Mutants-Light chain mutants with one cysteine at five different positions along the length of the polypeptide chain were expressed in high yield inE. coli (Table I). Expression and Characterization of LC2 Mutants-Light chain mutants with one cysteine at five different positions along the length of the polypeptide chain were expressed in high yield inE. By dissolving the aggregates in 6 M guanidine hydrochloride, most of the bacterial proteins were irreversibly denatured, whereas the light chains could be refolded and purified by ion-exchange chromatography (Saraswat and Lowey, 1991).The final yield of light chain was about 30 mg/liter culture. The engineered mutants described here differ both from the wild-type and recombinant LCs reported earlier (Saraswat and Lowey, 1991),in that they are missing one or both of the endogenous cysteines located near the COOH-terminal end of the native light chain. In order to compare the binding affinities of the mutant and native LCs, the light chains were exchanged into myosin in thepresence of EDTA at elevated temperatures (Fig. 1). The recombinant LCs can be distinguished from native LC2 by their slower mobilities
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