alphaB-crystallin maintains skeletal muscle myosin enzymatic activity and prevents its aggregation under heat-shock stress.
Here, we provide functional and direct structural evidence that alphaB-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 alphaB-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 alphaB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by approximately 90%, when myosin was thermally unfolded at 43 degrees C for 30 min, but was reduced by only approximately 42% when it was incubated with alphaB-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 alphaB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS alphaB-crystallin fluorescence studies confirmed the transient interaction of alphaB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that approximately 94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 degrees C for 30 min. alphaB-Crystallin, however, protected approximately 48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that alphaB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, alphaB-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.
Department of Biology and the Molecular Biology Institute, San Diego State University, 5550 Campanile Drive, San Diego, CA 92182-4614, USA.
Girish C
GC
Melkani
Anthony
A
Cammarato
Sanford I
SI
Bernstein
D000818:Animals; D052160:Benzothiazoles; D002645:Chickens; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D024744:Skeletal Muscle Myosins / Q000378:metabolism* / Q000648:ultrastructure; D013696:Temperature; D013844:Thiazoles; D038203:alpha-Crystallin B Chain / Q000378:metabolism* / Q000648:ultrastructure
D052160:Benzothiazoles; D006360:Heat-Shock Proteins; D013844:Thiazoles; D038203:alpha-Crystallin B Chain; C009462:thioflavin T; D024744:Skeletal Muscle Myosins