αB-Crystallin Maintains Skeletal Muscle Myosin Enzymatic Activity and Prevents its Aggregation under Heat-shock Stress

Girish C. Melkani, Anthony Cammarato, Sanford I. Bernstein

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)635-645
Number of pages11
JournalJournal of molecular biology
Volume358
Issue number3
DOIs
StatePublished - May 5 2006

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Keywords

  • electron microscopy
  • myosin II
  • protein aggregation
  • thermal unfolding
  • αB-crystallin

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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