Quaternary Structure and Dynamics of Polydisperse Molecular Chaperone Complexes

After their synthesis, the folding of proteins into their correct functional arrangements often requires assistance by 'molecular chaperones'. These proteins also play a vital role when the cell is stressed, by preserving and subsequently repairing damaged proteins. The mechanism by which these proteins carry out their 'housekeeping' duties is not fully understood, yet malfunctioning of this network results in a variety of disease states and even premature death. In this project we will focus on the least well understood family of the molecular chaperones, the Small Heat-Shock Proteins (sHSPs). In addition to their chaperone function, members of this protein family have been implicated in various disease states including cataract, motor neuropathy, and Alzheimer's disease.

Specifically, we will examine how sHSPs behave and interact with client proteins under stress conditions, and how they fit into the overall chaperone network in the cell. We have previously shown the sHSPs to be very dynamic, in that they can exchange subunits between each other on a rapid timescale. Their tendency to populate a range of states simultaneously, and to co-assemble with other sHSPs, has hampered conventional approaches to their study. We have focussed much of our previous research on developing and employing mass spectrometry (MS) strategies for the study of these proteins, and have developed a tool-kit well equipped for probing both their structure and dynamics.

The primary goal of this project is to examine in detail the complexes formed between the sHSPs and unfolding target proteins, which are the pivotal molecules in the sHSP chaperone pathway yet remain very poorly understood. We aim to characterise their organization, dynamic fluctuations, and their formation and disassembly. To achieve this ambitious target we will employ, and concomitantly develop further, a variety of MS-based approaches, including: the dissociation of the complexes in the gas phase; real-time reaction monitoring; and the direct measurement of their size.

To probe further the molecular details of these complexes between sHSP and target proteins we will perform molecular biology experiments in which we specifically mutate certain amino acids in the sHSPs to mimic modifications found in vivo. Thereby we can assess the effect of these alterations on the structure and dynamics of the complexes they form with substrate, allowing us to probe in detail the molecular mechanism of sHSP activity.

Therefore, through providing such insight into the way sHSPs interact with unfolding proteins we will go some way to understanding the pathway of their chaperone function. Furthermore, by clarifying their role in the overall molecular chaperone network, and examining the differences observed under conditions mimicking cellular stress, we hope to gain novel insight into the mechanism of their function.

The Small Heat-Shock Proteins (sHSPs) are a family of molecular chaperones and crucial components of the protein homeostasis network. Despite their importance they remain poorly understood, both functionally and structurally, due largely to their tendency to populate a polydisperse ensemble of inter-converting oligomers at equilibrium.

These dynamics are integral to their chaperone function, and consequently to study the sHSPs structural biology techniques are required which have a high resolution of separation in both spatial and temporal dimensions. Mass spectrometry (MS) has over the last few years evolved to become an excellent approach for the investigation of protein assemblies, providing details as to their subunit architecture, equilibrium fluctuations, and transient interactions.

Since the establishment of my independent research group we have employed MS to investigate the structure and dynamics of sHSPs from a range of organisms. Here we propose to extend this work to capitalise on two recent and very exciting breakthroughs we have made. Firstly we have demonstrated that we can identify the individual oligomers which comprise the wide range of complexes formed between sHSPs and their targets. Secondly, we have developed a means for determining likely structures adopted by polydisperse sHSPs. We propose to combine these methodological developments to elucidate the quaternary architecture and dynamics of the extremely heterogeneous sHSP:target complexes.

Our strategy will employ, and further develop, cutting-edge MS-based methods for structural biology. We will investigate wild-type mammalian sHSPs, mutants mimicking post-translational modification, and variants known to display aberrant chaperone activity. This project will reveal how sHSPs interact with client proteins, thereby providing a clearer view of their function and their role in proteostasis.

The proposed work will ultimately have practical importance within academia, industry, and have potential impact on human health. The research is directly applicable to four of BBSRC's strategic priority area: 'ageing research', 'systems approach to biological research', 'technology development for bioscience', and 'increased international collaboration'.

Ageing research: The protein homeostasis network of cell decreases in its efficiency during normal ageing, and can ultimately lead to a range of diseases. The sHSPs form a crucial component of this network, and therefore are candidates for pharmaceutical intervention. The work described here will provide insight into the pivotal complexes formed between sHSPs and their targets, and how they are regulated. This work will therefore be of immediate interest to companies and charities interested in the breakdown, and therapeutic re-establishment, of proteostasis.

Systems Approach to Biological Research: By providing insight into the structure and dynamics of the sHSPs and their interactions with targets, we will contribute of the 'systems view' of the cell. Similar to as described above, this will be important information for parties interested in how the cell maintains protein homeostasis. Furthermore there are interesting approaches to vaccine development being explored which rely on HSP:target complexes. Considering sHSPs are often major antigens (in M. tuberculosis for example), our characterisation of their interactions could potentially have considerable benefit for human health.

Technology Development for Bioscience: The technology and methodology we will employ, and further develop, in this project will be of wide applicability to structural biology in general. This is of direct interest to the MS manufacturers, whose market in the biosciences is continually expanding, and depend on breakthroughs into novel application areas.

Increased international collaboration: Our proposal includes collaboration with Prof Elizabeth Vierling at the University of Massachusetts, and the opportunity for the PDRA to visit her world-leading laboratories. This is in line with the BBSRC's encouragement of post-doctoral mobility and represents excellent training for the PDRA.