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.
Melkani
Girish C
GC
Department of Biology and the Molecular Biology Institute, San Diego State University, 5550 Campanile Drive, San Diego, CA 92182-4614, USA.
Cammarato
Anthony
A
Bernstein
Sanford I
SI
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