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Title: Biochemical studies on gelsolin : actin complexes and experiments to form a minimal, defined-length actin filament
Author: Wear, Martin Alexander
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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In this thesis we report the formation of a putative "capped-actin-minifilament" complex. This was created by combining the gelsolin:actin2 ternary (G:A2) and the actin:DNaseI binary (A:D) complexes together (1:1 molar ratio) under polymerising conditions (100mM KC1; 2mM MgC12 in the presence of 0.2mM CaC12). Size-exclusion data indicates the formation of a significantly larger species (in relation to G:A2), with an apparent stiochiometry of G:A3:D (gelsolin:actin:DNaseI, respectively). Kinetic and modelling evidence (Weber et al, 1994) suggests that the binding of two DNaseI molecules at the pointed-end of filaments is not possible due to a steric clash. Using DNaseI's ability to bind at the pointed-ends of actin monomers, we have probed the disposition of the monomers in the G:A2 complex. Size-exclusion, native-PAGE and fluorescence enhancement data (performed with NBD-Actin) indicate the formation of a stable, co-operative complex with a stoichiometry of G:A2:D2 (gelsolin:actin:DNaseI, respectively). The apparent Kd of A:D binding to the gelsolin:actin binary complex (G:A) is ~ 50nM, and is equivalent to the binding of G-Actin alone (Kd ~ 39nM). Our data are consistent with DNaseI having no effect on the interaction of actin monomers with gelsolin, and with the spatial orientation of monomers in G:A2 being different to those at the barbed-end of filaments. In contrast to this, data from fluorescence enhancement experiments with rhodamin-phalloidin (an actin filament specific binding molecule) provide evidence for the actin monomers, within the putative "minifilament", being in a filamentous-like conformation. We observe a specific binding, with significant levels of fluorescence enhancement (~ 3 - 4 fold), of rhodamine-phalloidin to the "minifilament", with an apparent Kd of ~4.6mM. We have also examined the possibility of replacing gelsolin (as the barbed-end capping protein) with a cloned polypeptide fragment derived from tensin, a component of focal adhesions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available