Studies on the cytoskeletal protein alpha-actinin.
thesisposted on 19.11.2015, 09:07 by Alistair. McGregor
The interaction between the cytoskeletal proteins ?-actinin and vinculin is considered to be fundamental to the link between actin microfilaments and the cytoplasmic face of integral membrane proteins that support cell adhesion. Evidence is provided here for an interaction between ?-actinin and vinculin. Electron microscopy of negatively stained samples was used to directly visualise ?-actinin/vinculin complexes. The micrographs showed that an ?-actinin dimer could bind two vinculin molecules, one at each end of the ?-actinin rod. Low speed equilibrium centrifugation demonstrated that the two proteins interacted with a Kd of approximately 1.6X10-5M. An interaction between ?-actinin and vinculin was also demonstrated using a 125I-vinculin overlay technique. Appropriate concentrations of unlabelled vinculin or ?-actinin inhibited the binding of 125I-vinculin to ?-actinin electroblotted onto nitrocellulose. The vinculin binding site of ?-actinin has been localised to a 37 amino acid sequence (residues 713 to 749). Thermolysin generated fragments of ?-actinin and glutathione-S-transferase linked ?-actinin fusion proteins were used to define the binding site. Their ability to bind vinculin was assayed using 125I-vinculin overlays, by their ability to compete in 125I-vinculin overlays, and in a solid phase binding assay. Determination of the three dimensional structure of ?-actinin may prove useful in elucidating a number of ?-actinin/protein interactions. As yet, intact ?-actinin has not been crystallised. The actin binding domain of ?-actinin (residues 1 to 268) was expressed in E. coli. A purification protocol that produces milligram quantities for crystallisation studies was devised. No crystals have as yet been produced. The repeats of ?-actinin (residues 240 to 749) were expressed. However, a satisfactory purification protocol could not be devised. The possible homology between the actin binding domains of ?-actinin and filamin was investigated. An immunological approach, in conjunction with peptide mapping, was used to identify the actin binding domain of filamin for N-terminal sequencing.