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Abstract
We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP). In in vitro expression assays, the GFP domain of the chimeric protein, S1(795)GFP, folds rapidly enabling us to monitor the folding of the motor domain using fluorescence. The myosin motor domain folds very slowly and transits through multiple intermediates that are detectable by gel filtration chromatography. The distribution of the nascent protein among these intermediates is strongly dependent upon temperature. At 25 degrees C and above the predominant product is an aggregate of S1(795)GFP or a complex with other lysate proteins. At 0 degrees C, the motor domain folds slowly via an energy independent pathway. The unusual temperature dependence and slow rate suggests that folding of the myosin motor is highly susceptible to off-pathway interactions and aggregation. Expression of the S1(795)GFP in the C2C12 muscle cell line yields a folded and functionally active protein that exhibits Mg(2+)ATP-sensitive actin-binding and myosin motor activity. In contrast, expression of S1(795)GFP in kidney epithelial cell lines (human 293 and COS 7 cells) results in an inactive and aggregated protein. The results of the in vitro folding assay suggest that the myosin motor domain does not fold spontaneously under physiological conditions and probably requires cytosolic chaperones. The expression studies support this conclusion and demonstrate that these factors are optimized in muscle cells.
Highlights
Muscle myosin, the prototype of the myosin II family, is a large asymmetric, multidomain protein composed of six polypeptides: two heavy chains of molecular weight 220,000 and two pairs of nonidentical light chains of molecular weights 17,000 –23,000
We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP)
We conclude that the folding of skeletal muscle myosin and the motor domain requires folding factors that are uniquely present in muscle cells
Summary
The core folding motif is a 7-strand, mostly parallel, -sheet that is flanked by three ␣-helices on each side and forms the ATP-binding pocket This structural motif is shared with the kinesin family of microtubule-based motors and with G-proteins involved in signal transduction [2, 3]. Myosin contains large insertions in the connecting loops of the core motif These insertions form an extended actin-binding surface and distinguish the myosin structure from kinesin and the G-proteins. We inferred in that study that folding of the myosin motor domain was rate-limiting and was mediated by cycles of chaperonin binding and release To confirm this hypothesis, we were interested in investigating the folding of the motor domain in the absence of the influence of the myosin light chains or dimerization of the heavy chain. We conclude that the folding of skeletal muscle myosin and the motor domain requires folding factors that are uniquely present in muscle cells
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