αB-crystallin maintains skeletal muscle myosin enzymatic activity and prevents its aggregation under heat-shock stress

Here, we provide functional and direct structural evidence that alpha B-crystallin, a member of the small heat-shock protein family, suppresses thermal unfolding and aggregation of the myosin II molecular motor. Chicken skeletal muscle myosin was thermally unfolded at heat-shock temperature (43 degrees C) in the absence and in the presence of alpha B-crystallin. The ATPase activity of myosin at 25 degrees C was used as a parameter to monitor its unfolding. Myosin retained only 65% and 8% of its ATPase activity when incubated at heat-shock temperature for 15 min and 30 min, respectively. However, 84% and 58% of the myosin ATPase activity was maintained when it was incubated with alpha B-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by similar to 90%, when myosin was thermally unfolded at 43 degrees C for 30 min, but was reduced by only similar to 42% when it was incubated with alpha B-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 degrees C for 30 min, while alpha B-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS alpha B-crystallin fluorescence studies confirmed the transient interaction of alpha B-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that similar to 94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 degrees C for 30 min. alpha B-Crystallin, however, protected similar to 48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that alpha B-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, alpha B-crystallin may be critical for nascent myosin folding, promoting myofibrillogenesis, maintaining cytoskeletal integrity and sustaining muscle performance, since heat-shock temperatures can be produced during multiple stress conditions or vigorous exercise. (c) 2006 Elsevier Ltd. All rights reserved.