In situ Studies of Structure Changes of Biomolecules at Interfaces and Under Fluidmechanical Stress

目的是更好地了解蛋白质变性和聚集的过程结构功能。调查将集中在蛋白质结构的变化和由此产生的聚集的生物分子的化学，热和（流体）机械应力条件的影响下。流体机械应力将在具有明确定义的流体机械应力分布的4辊装置中以及在不同几何形状的微流体通道中实现。原则上，变性可以发生在流体体积中或界面处。这种复杂的相互作用的表征需要一套全面的强大的分析方法。这将通过利用已建立的MWL-AUC在液相中进行蛋白质表征和通过ESI-DMA-MS在气相中进行蛋白质表征来实现。MWL-AUC通过对分离物质的沉降和紫外-可见吸收的组合分析提供独特的分辨率、准确度和重现性。此外，流体动力学和热力学蛋白质相互作用将进行详细研究。电喷雾电离耦合差分迁移率分析和质谱（ESI-DMA-MS）的新组合为气相中的蛋白质表征提供了很大的希望，从而能够研究摩尔质量高达1 MDa的蛋白质簇。将比较气相和液相中的方法的结果，并且它们的组合可以打开朝向形状分析的进一步途径。技术上相关的界面是空气-水界面（例如在泡沫中）和固体-水界面（例如颗粒或固体壁）。将利用手性非线性和频产生技术研究气-液和固-液界面上的蛋白质。用SFG法测定了各种蛋白质体系在气液界面的等电点，其结果与本体溶液的等电点基本一致。手性SFG将提供直接在界面和变性蛋白质结构的见解。将确定静电相互作用对水-空气界面和固-液界面处的吸附平衡和吸附动力学的作用，包括蛋白质-蛋白质相互作用和背景缓冲液的影响。

The objective is to better understand process-structure functions for protein denaturation and aggregation. The investigations will be focused on the changes of the protein structure and resulting aggregation of the biomolecules under the influence of chemical, thermal and (fluid-) mechanical stressing conditions. Fluid mechanical stressing will be realized in a 4-roll apparatus with well-defined fluid mechanical stress distributions and in microfluidic channels of varying geometry. In principle, denaturing can occur in the fluid volume or at interfaces. The characterization of this complex interplay requires a comprehensive set of strong analytical methods.This will be achieved by utilizing the established MWL-AUC for protein characterisation in the fluid phase and by ESI-DMA-MS in the gas phase. MWL-AUC offers unique resolution, accuracy and reproducibility by combined analysis of sedimentation and UV-Vis-absorption of separated species. Furthermore, hydrodynamic and thermodynamic protein interactions will be studied in detail. The novel combination of electrospray ionisation coupled to differential mobility analysis and mass spectrometry (ESI-DMA-MS) holds great promise for protein characterization in the gas phase enabling the investigation of protein clusters with molar masses up to 1 MDa. The results of the methods in the gas and liquid phase will be compared and their combination may open further pathways towards shape analysisTechnically relevant interfaces are air-water interfaces (e.g. in foams) and solid-water interfaces (e.g. particles or solid walls). Proteins at gas-liquid and solid-liquid interfaces will be studied by chiral nonlinear sum frequency generation (SFG). The isoelectric point (IEP) of various protein systems was determined by SFG at the gas-liquid interface and coincides mostly with the IEP in bulk solution. Chiral SFG will provide insights into the protein structures directly at the interface and on denaturation. The role of the electrostatic interactions on the adsorption equilibrium and adsorption kinetics at the water-air interface and the solid-liquid interface will be determined including the effects of protein-protein interactions and of the background buffer.