Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats

Whasil Lee, Johan Strümpfer, Vann Bennett, Klaus Schulten, Piotr E. Marszalek

Research output: Contribution to journalArticlepeer-review

Abstract

The conserved TPLH tetrapeptide motif of ankyrin repeats (ARs) plays an important role in stabilizing AR proteins, and histidine (TPLH)-to-arginine (TPLR) mutations in this motif have been associated with a hereditary human anemia, spherocytosis. Here, we used a combination of atomic force microscopy-based single-molecule force spectroscopy and molecular dynamics simulations to examine the mechanical effects of His → Arg substitutions in TPLH motifs in a model AR protein, NI6C. Our molecular dynamics results show that the mutant protein is less mechanically stable than the WT protein. Our atomic force microscopy results indicate that the mechanical energy input necessary to fully unfold the mutant protein is only half of that necessary to unfold the WT protein (53 versus 106 kcal/mol). In addition, the ability of the mutant to generate refolding forces is also reduced. Moreover, the mutant protein subjected to cyclic stretch-relax measurements displays mechanical fatigue, which is absent in the WT protein. Taken together, these results indicate that the His → Arg substitutions in TPLH motifs compromise mechanical properties of ARs and suggest that the origin of hereditary spherocytosis may be related to mechanical failure of ARs.

Original languageEnglish (US)
Pages (from-to)19115-19121
Number of pages7
JournalJournal of Biological Chemistry
Volume287
Issue number23
DOIs
StatePublished - Jun 1 2012
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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