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A MULTI-SPECTROSCOPIC APPROACH TO PROBE AMYLOID AGGREGATION AT BIOLOGICAL SURFACES

Periodic Reporting for period 1 - MultiSpecAMYLOID (A MULTI-SPECTROSCOPIC APPROACH TO PROBE AMYLOID AGGREGATION AT BIOLOGICAL SURFACES)

Reporting period: 2018-06-01 to 2020-05-31

Most neurodegenerative diseases, such as Alzheimer's and Parkinson's syndrome, are not curable, and only treatments managing the symptoms are available. The main cause of these diseases is the aggregation of protein and formation of insoluble fibrils, called amyloids, that interact with the cell's membrane and consequently cause cell damage. Many factors can trigger protein aggregation, e.g. overexpression of proteins, disturbance of protein clearance, or change in the membrane's composition. Although substantial knowledge has been acquired about protein aggregation, the cell membrane's influence on this process is poorly understood due to the lack of surface-specific techniques.

The overall aim of this project was to use novel spectroscopic techniques in order to understand the membrane's influence on protein aggregation. For this purpose, two techniques were used: surface-sensitive vibrational sum-frequency generation (VSFG) and multi-wavelength Raman spectroscopy, in combination with commonly used techniques such as atomic force microscopy (AFM), ThioflovinT fluorescence (ThT) and vibrational spectroscopy (FTIR). By combining all of these techniques, we provided relevant insights on how membrane interacts with protein aggregates. Firstly, we showed that the adsorption of protein aggregates is governed not only by electrostatic interaction, but hydrophobic interaction plays a substantial role as well. Hydrophobic interaction gets stronger when lipid monolayer has many defects, and some hydrophobic tails of the lipid are bent and even protruding water surface. We showed that due to hydrophobic interaction, aggregates are adsorbed to lipid monolayers when the amount of aggregates is so low, and we were are able to detect it only with VSFG. Secondly, we evaluated the influence of the lipid charge group on Abeta interaction with lipids. The composition of the living cell's membrane is very complex, so we chose two model lipids with different head groups: anionic and zwitterionic. Zwitterionic lipids are the main constituents of the cell's membrane, and we showed that adsorption of small aggregates, with β-hairpin-like structure, completely disintegrated the monolayer. Surprisingly, larger aggregates were adsorbed to the anionic monolayer, but it did not cause lipid monolayer damage. Finally, we applied multi-wavelength Raman spectroscopy to study protein aggregation and identified Amide band augmentation upon aggregation and shift of laser excitation line to shorter wavelengths. In addition, we found a spectral band that can be used as a marker for steric zipper formation.
Surface-sensitive vibrational sum-frequency generation spectroscopy (VSFG) combined with other techniques was applied to study the interaction between model system lysozyme and lipid monolayer and, afterward, was successfully applied to investigate Amyloid-beta, the protein that causes Alzheimer’s disease. Moreover, multi-wavelength Raman was used to gain molecular insights into protein aggregation.
Deliverable, four peer-reviewed publications, will result from the project and will be made Gold Open Access after the acceptance of publications. Various outreach activities were planned and taken in order to inform the general public about the project: a project’s web page, a presentation about Alzheimer’s, three popular articles, a cover illustration for Langmuir journal, posts in a social media and an occasional lab tours for students and others. For more details, please, see Technical Report Part B.
These results have a direct impact on the scientific community, which is very important for society as well. VSFG technique, which was employed in this project, is a relatively new technique, and for the first time, it was applied to study the formation and interaction of Aβ aggregates with lipid membranes. Hopefully, with more upcoming studies and scientific interest, VSFG will become a technique widely and routinely used for protein aggregation at liquid interfaces. Moreover, multi-wavelength Raman spectroscopy was employed, and findings broadened the possibilities to apply Raman spectroscopy for protein aggregation studies. The application of new techniques for protein aggregation studies will deepen our molecular knowledge and help us find ways to inhibit the protein aggregation.

For further information about this project and follow-up studies, please, check the project’s website:
www.surfacespectroscopy.com
The figure illustrates a surface specific technique VSFG and its application to study amyloids