Defining the mechanism of small molecule inhibition of amyloid fibril formation using ion mobility spectrometry-mass spectrometry

Amyloidosis is a pathological condition associated with the self-aggregation of proteins into highly ordered amyloid fibrils in vivo. Despite the importance of these high-profile disorders in today's ageing population and a wealth of on-going research, knowledge of the structural molecular mechanism of amyloid formation remains limited, primarily because of the complexity and heterogeneity within these systems, and rationally designed therapies are rare. Several small molecules have recently been found to modulate (stimulate or inhibit) the rate of fibrillogenesis of various amyloidogenic proteins in vitro but, in many cases, their reaction mechanism remains unknown. We have recently developed novel mass spectrometric methods to separate protein conformers and oligomers in real-time. Mass, kinetic and thermodynamic stability, and shape/cross-sectional area of each component within a heterogeneous assembly reaction can be measured in a single experiment using travelling wave ion mobility spectrometry coupled to mass spectrometry (IMS-MS; engineered by Micromass/Waters). Our data demonstrate the immense power of IMS-MS to resolve transiently populated species during the early stages of amyloidosis in vitro and form the basis for this project (Smith et al., J. Am. Soc. Mass Spectrom., 2007; Smith et al., PNAS, 2010). The aim of this project is to define amyloidogenic protein-ligand binding events in detail to pave the way to the rational design of ligands able to inhibit fibrillogenesis. To achieve this we will: (i) assess changes in the protein population (i.e. ratio of protein conformers, population of oligomers, etc.) caused by ligand presence; (ii) define which species bind the ligand; (iii) compare the conformational properties of the protein monomer and oligomers pre- and post-ligand binding; (iv) monitor the effect of ligand binding on the progress of fibril assembly. The student will initially study the protein-ligand binding characteristics of beta2-microglobulin, an amyloid-forming protein with which Ashcroft and Radford have gained much experience. Specifically, the student will study the rifamycin family of small molecule macrocycles, some of which we have found inhibit amyloid fibril formation whilst others have no effect. Using IMS-MS to analyse the mixture of protein conformers and oligomers, we will assess the changes in protein population resulting from ligand presence and identify ligand-binding species. Protein-ligand binding stability will be assessed from their binding constants and by MS/MS collision induced dissociation. To detect any conformational change on binding, the shape/cross-sectional areas of pre- and post-ligand binding protein species will be measured by IMS-MS. The protein regions involved in ligand binding will also be explored using novel HDX-IMS-MS methodology in collaboration with Micromass/Waters. The MS experiments will be complemented by other biophysical measurements (size exclusion chromatography, analytical ultracentrifugation) and the presence of fibrils in each experiment will be confirmed by electron microscopy. Protein mutants with different fibril-forming propensities which have been engineered by Radford will be compared to identify ligand binding residues. Non-amyloidogenic murine beta2-microglobulin will be used as a control. After establishing robust methods, the project will widen to encompass other proteins associated with amyloid diseases. Using the techniques described, their ligand binding properties will be investigated with known inhibitors. This will include the interactions of alpha-synuclein (Parkinson's disease) with baicalein and other flavanoids; Abeta (Alzhiemer's disease) with catechol derivatives; IAPP (type II diabetes) with resveratol. Thus, a correlation between ligand binding, protein conformation and fibril formation will be assessed which will ultimately pave the way for the rational design and screening of amyloid inhibition ligands.