TCB Publications - Abstract

Whasil Lee, Johan Strümpfer, Vann Bennett, Klaus Schulten, and Piotr E. Marszalek. Mutation of conserved histidines alters the tertiary structure and nanomechanics of consensus ankyrin repeats. Journal of Biological Chemistry, 287:19115-19121, 2012. (PMC: 3365944)

LEE2012 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 (HS). Here, we used a combination of Atomic Force Microscopy (AFM)-based single-molecule force spectroscopy and molecular dynamics (MD) simulations to examine the mechanical effects of H$\rightarrow$R substitutions in TPLH motifs in a model ankyrin repeat protein, NI6C. Our MD results show that the mutated protein is less mechanically stable than the wild-type (WT). Our AFM results indicate that the mechanical energy input necessary to fully unfold the mutated protein is only half of that necessary to unfold the WT protein (53 kcal/mol vs. 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. Taken together, these results indicate that the H$\rightarrow$R substitutions in TPLH motifs compromise mechanical properties of ARs and suggest that the origin of HS may be related to mechanical failure of ankyrin repeats.

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