Engineered cysteine mutants of myosin light chain 2. Fluorescent analogues for structural studies.
Site-directed mutagenesis has been used to insert cysteine residues at specific locations in the myosin light chain 2 (LC2) sequence. The aim was to modify these cysteines with one or more spectroscopic probes and to reconstitute myosin with labeled light chains for structural studies. Native LC2 has two endogenous cysteine residues at positions 126 and 155; a third sulfhydryl was added by replacing either Pro2, Ser73, or Pro94 with cysteine. By oxidizing the endogenous cysteines to an intramolecular disulfide bond (Katoh, T., and Lowey, S., (1989) J. Cell Biol. 109, 1549), it was expected that the new cysteine could be selectively labeled with a fluorescent probe. This proved more difficult to accomplish than anticipated due to the formation of secondary disulfide bonds between the newly engineered cysteines and the native ones. Nevertheless, the unpaired cysteines were labeled with 5-(iodoacetamido)fluorescein, and singly labeled species were purified by ion-exchange chromatography. Chymotryptic digestion of the light chains, followed by high performance liquid chromatography separation of the peptides, led to the identification of the fluorescein-labeled cysteines. After light chain exchange into myosin, the position of the thiols was mapped by antifluorescyl antibodies in the electron microscope. Rotary-shadowed images showed the antibody bound at the head/rod junction of myosin for all the mutants. These mapping studies, together with the finding that widely separated cysteines can form multiple disulfide bonds, support a model for LC2 as a flexible, globular molecule that resembles other Ca/Mg-binding proteins in tertiary structure.
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
L D
LD
Saraswat
S
S
Lowey
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D002645:Chickens; D002853:Chromatography, Liquid; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005818:Genetic Engineering; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009218:Myosins / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure; D010084:Oxidation-Reduction; D010957:Plasmids; D011487:Protein Conformation
D000906:Antibodies; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009218:Myosins; D003545:Cysteine; D019793:Fluorescein