The mechanism of a multi-chaperone system for promoting protein disaggregation

Proteins are the work-horses of the cell performing or catalysing many of the processes that are essential for life. In order to perform their appropriate function proteins, which are composed of a linear chain of amino acids, must fold up into the correct three-dimensional structure in the correct place within the cell, or be targeted for destruction when damaged. To achieve this a set of proteins have evolved, known as molecular chaperones, whose function is essentially one of protein quality control, ensuring proteins fold correctly or are unfolded and targeted for degradation. One particularly acute problem is when the cell is exposed to harsh environmental conditions, such as heat, cold or chemical insult. This causes proteins to misfold and self-associate to form insoluble aggregates. These aggregates can cause cell-death and in some cases disease e.g. CJD, Alzheimer's disease. The protein to be studied in this project, ClpB, is a bacterial ATP-driven motor protein (part of the so-called AAA ATPase family) which is able to rescue proteins which have previously aggregated, thus helping the cell to recover. It adopts a ring structure and rescues proteins by translocating them through its central pore in a process dependent on the energy provided by ATP turnover. The mechanism is also analogous to that seen in similar AAA ATPase proteins which are involved in a diverse set of cellular functions. The aims of the proposed work is to investigate exactly how the energy derived from ATP is coupled to protein translocation through the ClpB protein complex. This involves measuring the nucleotide binding and hydrolysis characteristics in the two ATP binding sites on each polypeptide and evaluating how these two sites communicate with each other. The kinetics of the structural changes in the protein induced by the binding and hydrolysis of ATP, as well as those of the protein substrate being translocated will determined and compared. We are also going to examine the aggregation and amyloidogenesis processes at the level of single molecules (or single particles) to determine exactly which aggregates can be dissolved by molecular chaperones, and exactly how this is achieved.

The molecular chaperone ClpB is part of a bi-chaperone system which, along with DnaK/DnaJ/GrpE (KJE), has the unique ability to rescue proteins from an aggregated state. It is also a member of the AAA+ protein superfamily whose members are ATPases involved in a diverse set of cellular processes. Like members of that family, ClpB has two nucleotide binding sites and assembles into a hexameric ring with a pore running through the centre of the complex. It also has a unique coiled-coil domain, which lies between the two nucleotide binding domains, and protrudes laterally from the ring. Disaggregation is achieved as the DnaK system disrupts a protein substrate from the aggregate and then transferred it to ClpB where it is translocated through the central pore in an ATP-dependent manner before it can refold to its native state. Although the basic details of this mechanism are known there is, as yet, no quantitative, kinetic or structural description of how ClpB couples the energy derived from ATP hydrolysis to protein translocation through its central pore. The work proposed here aims to elucidate the mechanism of this molecular motor by (a) determing the kinetics of ATP-induced conformational changes in ClpB and how they are coupled to binding, translocation and disaggregation of polypeptide substrates; (b) using single-molecule fluorescence burst analysis spectroscopy to examine the heterogeneity of the aggregation process, both for amorphous aggregates and amyloid fibrils, and determine which sub-population can be reversed by either KJE or KJE and ClpB ; (c) determining the precise nature of the interaction between DnaK and ClpB. This will be achieved principally using a variety of fluorescence techniques, (e.g. intensity and FRET) in both ensemble and single-molecule experiments.