Abstract
Structural analyses of actin binding regions comprising tandem calponin homology domains alone and when bound to F‐actin have revealed a number of different conformations with calponin homology domains in ‘open’ and ‘closed’ positions. In an attempt to resolve these issues we have examined the properties of the utrophin actin binding domain in open and closed conformations in order to verify the conformation when bound to F‐actin. Locking the actin binding domain in a closed conformation using engineered cysteine residues in each calponin homology domain reduced the affinity for F‐actin without affecting the stoichiometry furthermore differential scanning calorimetry experiments revealed a reduction in melting temperature on binding to actin. The data suggest the amino‐terminal utrophin actin binding domain is in an open conformation in solution and when bound to F‐actin.