In-Situ Investigation of Structural Changes of Biomolecules at Interfaces and under the Influence of Fluid Mechanical Stress

The integrity of proteins is a decisive factor in their functionality. However, during production and processing, the flexible biomolecules are frequently exposed to external mechanical stress and interact with different surfaces, both might affect their folding. Structural changes promote aggregation induced by hydrophobic interactions. Stress-treated, genetically modified beta-lactoglobulin (BLG) proteins have been investigated in the bulk phase by analytical ultracentrifugation (AUC) and directly at the water/air and solid/water interfaces by sum-frequency generation (SFG) spectroscopy. The influence of the solvent pH, salt concentration or addition of denaturating agents on the protein structural changes and the resulting particle-particle interactions have been investigated revealing the important role of electrostatic interactions. Interfacial isoelectric points (IEP) of various proteins such as BLG, bovine serum albumin (BSA), hemoglobin (Hem), lysozyme (Lys), insulin, and a number of others have been measured by surface-sensitive SFG spectroscopy and found to be almost identical to that in the bulk phase. Chiral SFG measurements indicate that proteins at the interface can be less susceptible to structural changes than in the liquid phase. Electrospray ionization mobility–classified mass spectrometry (ESI-DMA-MS) has been additionally used to characterize protein size, shape, and density in the gas phase via single-molecule pycnometry.