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Molecular mechanism of a group II chaperonin.

Reference	BB/F007345/1
Principal Investigator / Supervisor	Professor Anthony Clarke
Co-Investigators / Co-Supervisors
Institution	University of Bristol
Department	Biochemistry
Funding type	Research
Value (£)	414,455
Status	Completed
Type	Research Grant
Start date	01/03/2008
End date	31/08/2011
Duration	42 months

Abstract

The genes encoding the thermosome and its co-protein, prefoldin (PFD), have been cloned and over-expressed in E.coli in my laboratory, thus providing ideal study objects for elucidating the mechanism of the biologically vital but poorly understood type II chaperonins. The thermosome comprises 16 subunits of two homologous types (alpha & beta), which form an alpha8/beta8 structure. Both types of subunit are ATPases and the binding and hydrolysis of nucleotides is used to drive rearrangements of the structure. The project has the following experimental angles. (a) Mutagenesis will be used to produce two types of ATPase variants; those in which ATP hydrolysis is blocked and those in which binding is blocked altogether. These mutations will be made in the alpha and beta subunits individually and crossed over to define the interplay between subunits in the ATPase cycle. (b) Steady-state kinetics will be used to define the overall reaction mechanism with respect to the binding and hydrolysis of ATP and to assess the influence of the nucleotide-site mutations. (c) The wild-type and ATPase mutant forms will be studied by transient kinetic techniques, using quenched-flow to monitor the chemistry of ATP turnover and product release and stopped-flow to measure conformational shifts reported by the single tryptophan residue in the alpha subunits. This information will be augmented by monitoring responses in the beta subunit by use of an engineered single-tryptophan version of the chaperonin. (d) Heat-stable enzymes will be used as substrates to look at substrate binding, folding and ejection dynamics during the ATPase cycle. (e) Labelled PFD will be used for optical measurements of co-protein behaviour (f) Binding surfaces on the chaperonin for PFD and substrate will be probed by mutagenesis and the measurement of equilibrium and transient binding affinites.

Summary

All the major functions of the cell are performed by proteins these include movement, the generation of energy, growth, division and signalling. However, as the breadth and complexity of these tasks might suggest, protein molecules are necessarily structurally complex. The sequence of each protein chain is dictated by the gene that encodes it, but until and unless these chains fold up into precise shapes they have no biological activity. The machinery that makes the chain is called the ribosome and the growing chain is extruded in an unstructured state that must then fold and assemble to its biologically active structure after it is released. It is at this point that the cell employs a group of helper proteins, known as molecular chaperones, the function of which is to assist the process of folding and assembly. Molecular chaperones are themselves protein molecules and take different forms depending on their role. Some are small, inert structures that coat unfolded protein chains, while at the other end of the spectrum there are large energy-requiring machines that perform complex tasks. One of the latter classes is termed the chaperonins which are cavern-like, toroidal structures capable of capturing unfolded protein chains and sequestering them in a central cavity. When in this environment the chain can spontaneously fold into a shape dictated only by the sequence of amino acids and driven by the chemical properties of these constituent units. The necessity for the folding process to occur in this capsule arises from the molecular properties of unfolded protein chains. In the folded, biologically active state, the hydrophobic (water-avoiding) amino acids are clustered in the core to minimise contact with water and the structure is compact; indeed it is the burial of these groups that drive folding. In its newly synthesised state, prior to folding, hydrophobic amino acids are exposed; this causes aggregation of protein chains which are inactive. By sequesteringindividual unfolded chains and allowing them to fold in isolation, this potential aggregation process is avoided and folding is highly efficient. The workings of a device on the tens of nanometre scale that can do these things are intriguing. The machine must be able to bind to the unfolded protein chain, swallow it into a cavity, close the cavity to allow folding in a protected space, re-open and eject the folded protein into the cellular environment where it can perform its biological function. To do this chaperonins require chemical energy in the form of ATP hydrolysis; i.e. they are chemo-mechanical devices capable of turning chemistry into molecular movements. Evolution has produced two designs for these folding machines; the group I chaperonins found in bacteria, chloroplasts and mitochondria and the group II which are found everywhere else, i.e. in the cytoplasm of all eukaryotes and in archaea. The group I version is reasonably well understood at the level of its chemically driven mechanism, but the group II chaperonins are more mysterious and more challenging to study. The focus of the work we propose is a group II chaperonin known as the Thermosome. Although we know the structure of this complex protein, its mechanism is ill-defined and our aim is to elucidate the mechanical states through which this device is driven as it goes through the chemical cycle of ATP hydrolysis. By employing optical methods which report motions within the Thermosome and which are sensitive to molecular docking events, we will determine how the processes of binding a protein substrate, encapsulating it and then releasing it is driven and orchestrated by the chemical events in the ATP sites of this complex molecule. Not only will this contribute to our knowledge of cellular function, but in the long-run an understanding of the workings of biologically evolved molecular devices will be invaluable in the design and synthesis of man-made nano-machines.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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