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

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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.