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

目的是为了更好地理解蛋白质变性和聚集的过程结构功能。研究的重点将集中在蛋白质结构的变化和生物分子在化学、热和(流体)机械应力条件下的聚集。流体机械应力将在具有明确流体机械应力分布的四辊装置中实现，并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或界面上。对这种复杂相互作用的表征需要一套全面的强有力的分析方法，这将通过利用建立的MWL-AUC在液体相中表征蛋白质和通过ESI-DMA-MS在气相中表征来实现。MWL-AUC通过对分离物种的沉降和UV-Vis吸收进行联合分析，提供了独特的分辨率、准确性和重现性。此外，还将详细研究流体力学和热力学蛋白质之间的相互作用。电喷雾电离与差示迁移率分析和质谱仪(ESI-DMA-MS)的新组合为气相中的蛋白质表征带来了巨大的希望，使研究摩尔质量高达1 mda的蛋白质簇成为可能。技术上相关的界面是空气-水界面(例如泡沫)和固体-水界面(例如颗粒或固体壁)。利用手征非线性和频(SFG)方法研究了气液界面和固液界面的蛋白质。用SFG测定了不同蛋白质体系在气液界面的等电点(IEP)，并与本体溶液中的等电点基本一致。手性SFG将直接提供对蛋白质界面结构和变性结构的洞察。静电相互作用对水-气界面和固-液界面吸附平衡和吸附动力学的作用将被确定，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.