Kinetic studies of actin folding by the chaperonin CCT

• Main Author: Stuart, Sarah Frances
• Other Authors: Leatherbarrow, Robin ; Matthews, Steve ; Willison, Keith
• Published: Imperial College London 2012
• Subjects: 571.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556569

The chaperonin containing TCP1 (CCT) is an essential protein in eukaryotes, and is vital for the correct folding of various obligate substrates including the cytoskeletal proteins actin and tubulin and the WD40 repeat containing proteins Cdc20 and Cdh1. CCT is highly complex and interacts with some proteins only transiently; as such the study of the mode of binding and the mechanism by which proteins are folded by the molecular chaperone is very challenging. I have used spectroscopic techniques to study the folding of Cdh1 and actin by CCT. The dimensions of CCT are such that Förster resonance energy transfer (FRET) could be used to measure distances within the complex, providing that fluorophores could be selectively introduced. Through labelling of a fragment of the WD40 propeller of Cdh1 and binding of a fluorescently labelled calmodulin (CaM) to the calmodulin binding peptide (CBP) tag introduced into two CCT subunits, it was hoped that subunits which bind to this region of Cdh1 could be identified. Following expression and purification of a stable Cdh1 fragment, methods for selective labelling of the protein were developed. The complex formed between the fluorescently labelled Cdh1 fragment, CaM and CCT was isolated by sucrose gradient. Unfortunately, no significant, reproducible FRET signal could be identified, indicating that this technique was not suited to the study of Cdh1 folding by CCT. Actin is an essential eukaryotic protein which polymerises to form filaments involved in many different cellular processes. CCT subunits which interact with actin have been identified, but the mechanism by which actin is folded is not well understood and has previously relied on the trapping of actin-CCT complexes from non-functional systems. Since the discovery of PLP2 as a cofactor involved in the actin folding process in yeast, pure in vitro actin assays have been possible. Through labelling of the C-terminus of actin with the environmentally sensitive fluorophore acrylodan, it was possible to monitor the kinetics of actin folding by CCT spectroscopically. Non-hydrolysable ATP analogues were used which allowed the identification of two separate stages in the actin folding process on CCT, an initial rearrangement of the two domains of actin followed by packing of the actin C-terminus. The importance of the actin C-terminus in actin folding and stability was highlighted by the effect of the probe with which actin was labelled on folding. The effect of mutations in CCT on the rate of actin folding was also investigated using the spectroscopic folding assay, as well as the effect of truncations of PLP2. It was found that the mutant CCTanc2, which is from a strain of yeast that produces defective actin structures and contains a mutation in subunit CCT4, behaves differently to wild type CCT at higher temperatures and concentrations of ATP. PLP2 appears to be exceptionally important for productive actin folding by CCTanc2.