Understanding the relationship between aggregating proteins, mis-folding disease and molecular chaperones

A growing number of human disorders including Alzheimer’s disease (AD) and Parkinson’s disease (PD), type II diabetes and the prion diseases are associated with the deposition of proteinaceous ‘amyloid’ aggregates in human tissues. The incidence of these conditions is increasing as populations age, with an associated national cost to the UK alone larger than both cancer and heart disease combined.
All is not lost, however! A set of proteins is up-regulated 
in vivo in response to the presences of such proteinacous aggregates. In vitro, these same proteins are able to dramatically reduce the tendency of proteins to aggregate. The primary focus of the lab is to better understand the mechanism by these proteins inhibit, and in some cases prevent, protein aggregation and amyloid formation.

NMR Spectroscopy: structure and dynamics at atomic resolution

Biological organisms rely on proteins to perform the majority of the chemical tasks needed to sustain life. All functions that proteins perform stem from the three-dimensional structures that they adopt. Because these structures are stabilized by weak non-covalent interactions, at temperatures relevant to biology they are easily rearranged by thermal motion. Proteins are consequently very dynamic molecules that are best understood as an ensemble of inter-converting conformers, rather than single static structures.

The majority of experimental techniques available for structural biology however, report on the ‘average’ positions of the constituent atoms. In order to fully understand how biological molecules function, it is necessary to move beyond static structures. NMR spectroscopy is unique in that it can simultaneously describe the motions that proteins undergo as well as their structure. It can therefore yield great insight into how proteins ‘work’.

There are two major types of experiments that we employ. The first, termed here as ‘fast’ measurements (above, green), are able to characterise motion on the pico/nano second timescale. In structured proteins, these relatively rapid motions include bond liberations, and side chain rotations. In unfolded proteins, these motions can be more extensive, reflecting the inter-conversion of many disordered conformations.

Solution NMR spectroscopy is often considered to have an inherent size limit of proteins only a few tens of kilo-Daltons in mass. However, exploiting the detailed spin physics and preparing carefully labelled samples, we are able to routinely perform both fast and slow dynamical measurements on proteins up to the mega-Dalton size range. To put this in context, without these improvements, approximately 10% of single chain proteins within the human genome are within the sights of solution NMR. With these improvements, 99% of the single chain proteins, and many of their complexes can potentially be analysed using solution NMR. When looking at complexes larger than 1MDa, we also employ solid state NMR techniques.

Translating atomic resolution information onto the oligomer level with ion-mobility mass spectrometry and electron microscopy

The NMR experiments described above provide ‘local’ information about atomic structure and dynamics. We combine these data with measurements from ion-mobility mass spectrometry, which is able to give us three important bits of additional information: which oligomeric states are populated in solution, the timescale on which any quaternary structure rearranges and even the dimensions of individual oligomers, without the need for prior separation. By combining the local structural restraints derived from NMR, with the lower resolution but oligomer specific ‘global’ measurements, we are able to obtain a very rich picture of macromolecular behaviour.