Mapping single cysteine mutants of light chain 2 in chicken skeletal myosin.
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.
Saraswat
L D
LD
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
Pastra-Landis
S C
SC
Lowey
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000648:ultrastructure
D000936:Antigen-Antibody Complex; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009218:Myosins; D003545:Cysteine