TY - JOUR
T1 - αB-Crystallin Maintains Skeletal Muscle Myosin Enzymatic Activity and Prevents its Aggregation under Heat-shock Stress
AU - Melkani, Girish C.
AU - Cammarato, Anthony
AU - Bernstein, Sanford I.
N1 - Funding Information:
This work was supported by Muscular Dystrophy Association research grant 3682, NIH grant R01 AR43396 and NIH grant R01 GM32443 to S.I.B., NIH research supplement AR43396-S1 to A.C. and NSF equipment grant DBI-030829 to Dr Steven Barlow. We thank Dr Jose A. Mendoza (California State University, San Marcos) for allowing us to use his spectrofluorimeter and Dr Roger Craig (University of Massachusetts Medical School) for helpful suggestions and discussions concerning sample preparation and analysis by electron microscopy. We thank Dr Aileen Knowles (San Diego State University) for helpful experimental suggestions and comments on the manuscript.
PY - 2006/5/5
Y1 - 2006/5/5
N2 - Here, we provide functional and direct structural evidence that αB-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 °C) in the absence and in the presence of αB-crystallin. The ATPase activity of myosin at 25 °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 αB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by ∼90%, when myosin was thermally unfolded at 43 °C for 30 min, but was reduced by only ∼42% when it was incubated with αB-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 °C for 30 min, while αB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS αB-crystallin fluorescence studies confirmed the transient interaction of αB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that ∼94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 °C for 30 min. αB-Crystallin, however, protected ∼48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that αB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, αB-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.
AB - Here, we provide functional and direct structural evidence that αB-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 °C) in the absence and in the presence of αB-crystallin. The ATPase activity of myosin at 25 °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 αB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by ∼90%, when myosin was thermally unfolded at 43 °C for 30 min, but was reduced by only ∼42% when it was incubated with αB-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 °C for 30 min, while αB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS αB-crystallin fluorescence studies confirmed the transient interaction of αB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that ∼94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 °C for 30 min. αB-Crystallin, however, protected ∼48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that αB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, αB-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.
KW - electron microscopy
KW - myosin II
KW - protein aggregation
KW - thermal unfolding
KW - αB-crystallin
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U2 - 10.1016/j.jmb.2006.02.043
DO - 10.1016/j.jmb.2006.02.043
M3 - Article
C2 - 16546210
AN - SCOPUS:33646042860
SN - 0022-2836
VL - 358
SP - 635
EP - 645
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 3
ER -