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Title: Characterisation of the actin-binding site of alpha-actinin.
Authors: Kuhlman, Philip Alan.
Award date: 1993
Presented at: University of Leicester
Abstract: The alpha-actinin residues involved in the interaction of chicken smooth muscle alpha-actinin with F-actin were defined by co-sedimentation. Residues 120 to 140 of alpha-actinin were identified as essential with residues 141 to 159 and 1 to 107 contributing to the interaction. Temperature dependent binding and the lack of inhibition by high salt concentrations implicated hydrophobic interactions in the binding mechanism. Further characterisation of the binding interaction using stopped-flow techniques determined a K for the interaction of 0.82 muM at 15°C and 2.38 muM at 25°C. These techniques distinguished between the initial binding event and the subsequent bundling of the actin filaments. The consequences of bundling on the binding equilibrium are discussed. The role of the actin N-terminus as the initial docking site for alpha-actinin has been investigated using a peptide corresponding to this region of actin. The lack of any detectable interaction suggests that this region of actin does not interact with alpha-actinin. This is consistent with the mapping of the binding site for alpha-actinin on the opposite side to the N-terminus of the actin molecule. In addition to studying the binding mechanism the structure of the alpha-actinin actin-binding domain (ABD) was investigated. This domain of alpha-actinin is proposed to consist of two smaller sub-domains. Attempts to crystallise the ABD of alpha-actinin have failed due to the amphipathic nature of the domain. The alpha-actinin interaction with F-actin is proposed to be mediated by both electrostatic and hydrophobic forces. The interaction site is composed of distinct sites within the primary sequence. This model is similar to those proposed for the interaction of DNase I, myosin and gelsolin segment 1 with actin.
Level: Doctoral
Qualification: Ph.D.
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, Dept. of Biochemistry
Leicester Theses

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