Investigating the mechanism of hydrodynamic force induced protein aggregation

We have shown recently that extensional flow is damaging to proteins and that it can induce protein aggregation. The pathway of extensional flow induced unfolding and the mechanism of aggregation is, however, still unknown. Understanding how extensional flow leads to aggregation would ultimately allow rational control strategies to be established for overcoming flow-induced aggregation. To be able to achieve this goal it is necessary to establish a quantitative analytical or numerical model of the aggregation process, akin to those derived previously for protein aggregation in the absence of flow. The aim of this studentship is to develop such a model. This will be achieved by:
(i) understanding how the properties of the protein (sequence, architecture, concentration, size/topology and solution conditions (pH etc)) affect the probability of protein aggregation under flow.
(ii) understanding how the properties of the flow field (strain rate, shear rate, shear length and pass number) affect the probability of protein aggregation under flow.
(iii) mapping and quantifying the extent of flow-induced unfolding. This will be achieved using both mass spectrometric and single molecule fluorescence (SMF) methods. The latter method will be achieved by site specifically labelling the protein under study with a donor and acceptor fluorophore and performing FRET experiments in situ during extensional flow. This approach will allow the extent of unfolding during extensional flow (and, importantly, the degree of refolding after exiting this region) to be quantified. To map the unfolding pathway for model and bio-pharmaceutically relevant proteins in residue-specific detail, we will employ hydrogen/deuterium exchange (HDX) monitored by mass spectrometry. Positions of exchange as a function of exposure time will then be determined by immediate pepsin digestion (on column) and LC-MSMS (using ETD for sequencing) and compared with protein under quiescent conditions, revealing force-labile regions. These data will be correlated with the measured aggregation rate and aggregation propensity of the exposed region (using TANGO and SOLUBIS) to link flow-induced exposure of particular regions of the polypeptide chain to the onset of aggregation.