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Title: Single molecule studies on actin folding by the eukaryotic chaperonin CCT
Author: Seaby, Thomas David
ISNI:       0000 0005 0733 6137
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
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The eukaryotic, cytosolic chaperonin containing TCP-1 (CCT) is a protein folding nanomachine. CCT is a 1 MDa double-ring-structured ATPase which assists nascent polypeptides to reach their native conformational state. Obligate substrates include the cytoskeletal components actin and tubulin. Whilst significant structural studies have been conducted, relatively little is known about the dynamics of the ATP-dependent folding process. Total Internal Reflection Fluorescence (TIRF) microscopy is a high signal to noise, single molecule, microscopy technique which has been previously used to observe both protein dynamics and extract enzyme kinetics at the single molecule level. We have immobilised functional CCT onto silica surfaces in order to extract dwell times and kinetics of the folding process, as CCT captures, folds and releases actin from its unfolded state to the folded G-actin monomer. A method for the immobilisation of CCT, via a calmodulin protein, has been developed. Mutant CCT-6CBP has a calmodulin binding peptide tag inserted into the apical domain of the CCT6 subunit and can be affinity captured via calmodulin immobilised to a borosilicate surface. The calmodulin-CCT interaction is calcium dependent and can be reversed allowing for discrimination between specific versus non-specific binding. ATP-dependent release of unfolded actin from CCT complexes immobilised to silica surfaces has proven to be problematic. Complexes can be specifically bound but subsequently seem to lose functional folding behaviour. Attempts to count the numbers of the stimulatory cofactor phosducin-like protein 2 (PLP2) and actin monomers bound to the CCT complex, utilising photobleaching, is described. A program for the analysis of single molecule traces was written. Incorporating a Chung-Kennedy non-linear filter, this program provides stoichiometric data consistent with spectrophotometric methods. Analysis of CCT-actin-PLP2 complexes was not possible due to time constraints.
Supervisor: Klug, David; Willison, Keith Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral