Investigating the mechanism of hydrodynamic force induced protein aggregation

我们最近的研究表明，拉伸流对蛋白质具有损伤作用，并能诱导蛋白质聚集。然而，伸展流诱导展开的途径和聚集的机制尚不清楚。了解外延流动是如何导致聚集的，将最终允许建立合理的控制策略来克服流动引起的聚集。为了能够实现这一目标，有必要建立一个聚集过程的定量分析或数值模型，类似于先前导出的没有流动的蛋白质聚集模型。这次学习的目的就是发展这样一个模式。这将通过：(i)了解蛋白质的性质（序列、结构、浓度、大小/拓扑和溶液条件（pH等））如何影响蛋白质在流动下聚集的概率来实现。（ii）了解流场的性质（应变速率、剪切速率、剪切长度和通过数）如何影响流动下蛋白质聚集的概率。（iii）绘制和量化流动引起的展开程度。这将通过质谱和单分子荧光（SMF）方法来实现。后一种方法将通过用供体和受体荧光团对所研究的蛋白质进行定点标记，并在拉伸流动中进行原位FRET实验来实现。这种方法将允许在伸展流动期间展开的程度（重要的是，退出该区域后的再折叠程度）被量化。为了在残基特异性细节中绘制模型和生物药学相关蛋白的展开途径，我们将采用质谱监测的氢/氘交换（HDX）。然后，交换位置作为暴露时间的函数将通过即时胃蛋白酶消化（柱上）和LC-MSMS（使用ETD测序）确定，并与静止条件下的蛋白质进行比较，揭示力不稳定区域。这些数据将与测量的聚集率和暴露区域的聚集倾向（使用TANGO和SOLUBIS）相关联，以将多肽链特定区域的流动诱导暴露与聚集的发生联系起来。

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.