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

界面处和流体机械应力下生物分子结构变化的原位研究

• 批准号: 315396049
• 负责人: Professor Dr.-Ing. Wolfgang Peukert
• 金额: --
• 依托单位: Lehrstuhl für Feststoff- und Grenzflächenverfahrenstechnik (LFG)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/315396049?language=en
• 关键词: situ; Studies; Structure; Changes; Biomolecules

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.

目的是更好地了解蛋白质变性和聚集的过程结构功能。研究将集中在化学、热和（流体）机械应力条件下蛋白质结构的变化和生物分子的聚集。流体力学应力将在具有明确流体力学应力分布的四辊装置和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处。描述这种复杂的相互作用需要一整套强有力的分析方法。这将通过在液相中利用已建立的MWL-AUC进行蛋白质表征，在气相中利用ESI-DMA-MS来实现。MWL-AUC通过对分离物种的沉淀和紫外-可见吸收的综合分析，提供了独特的分辨率、准确性和再现性。此外，还将详细研究流体力学和热力学蛋白质相互作用。电喷雾电离耦合差分迁移率分析和质谱（ESI-DMA-MS）的新组合在气相蛋白质表征方面具有很大的前景，能够研究摩尔质量高达1mda的蛋白质团簇。将比较气相和液相方法的结果，它们的组合可能为形状分析开辟进一步的途径。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）。用手性非线性和频率生成（SFG）方法研究气液界面和固液界面上的蛋白质。不同蛋白质体系的等电点（IEP）是用SFG法测定的，与本体溶液中的等电点基本一致。手性SFG将提供直接在界面上的蛋白质结构和变性的见解。静电相互作用对水-空气界面和固-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。