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Structural and functional impact of site-directed methionine oxidation in myosin

Jennifer C Klein1, Rebecca J Moen, Evan A Smith

  • 1Department of Chemistry, Saint Olaf College, Northfield, Minnesota 55057, United States.

Biochemistry
|October 13, 2011
PubMed

Insights

Site-directed methionine oxidation in Dictyostelium myosin II reveals M394 is sensitive to oxidation, impacting actomyosin function. This oxidation causes structural changes in the actin-binding cleft, relevant to aging and disease.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Protein oxidation is crucial for cellular function but implicated in aging and disease.
  • Oxidative stress can impair muscle function, linked to actomyosin dysfunction.
  • Understanding molecular mechanisms of protein oxidation is vital for disease and aging research.

Purpose of the Study:

  • To investigate the structural and functional consequences of methionine oxidation in Dictyostelium myosin II.
  • To identify specific methionine residues sensitive to oxidation and their impact on actomyosin interactions.
  • To correlate molecular-level oxidation effects with observed muscle dysfunction.

Main Methods:

  • Site-directed mutagenesis to create methionine-to-leucine variants.
  • In vitro oxidation using peroxide.
  • Site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy.
  • Measurement of actin-activated myosin ATPase activity.

Main Results:

  • Peroxide treatment reduced actin-activated myosin ATPase activity in native Dicty myosin II.
  • Methionine-to-leucine mutations identified M394 as a key oxidation-sensitive site near the actin-binding interface.
  • EPR studies showed oxidation altered spin-label mobility and distances in the actin-binding cleft, especially with actin present.
  • Overall protein secondary structure and thermal stability remained unaffected by oxidation.

Conclusions:

  • Oxidation-induced structural changes in myosin II involve a redistribution of conformational states within the actin-binding cleft.
  • Methionine oxidation, particularly at M394, directly impacts actomyosin function.
  • These findings provide molecular insights into protein oxidation's role in muscle dysfunction, aging, and disease.

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