(534e) Effect of Oscillating Electric Field on Preformed Human Serum Albumin Fibrils

Authors: 
Sen, S., Indian Institute of Technology Kharagpur
Chakraborty, M., Indian Institute of Technology Kharagpur
Goley, S., Indian Institute of Technology Kharagpur
Dasgupta, S., Indian Institute of Technology Kharagpur
DasGupta, S., Indian Institute of Technology Kharagpur

The stability and biological activity of proteins are highly correlated to their three dimensional structures with multi-level conformations. In the native folded state, proteins are soluble in aqueous solution. When the structure deviates from the native state, proteins have a propensity to aggregate. Such deviation can occur when proteins are subjected to certain specific biophysical conditions, where they tend to undergo self-aggregation to form polymeric species with different morphologies leading to the formation of insoluble amyloid fibrils. The extensive depositions of fibrils, associated with high cross β-sheet content in extracellular spaces of various tissues causing cellular damages, are linked to a number of neurological disorders such as Alzheimer’s, Parkinson’s, Creutzfeldt-Jakob disease, type II diabetes etc.[1] The possible remedy is removal of these toxic fibrillar deposits from affected tissues by developing inhibitors or disintegrating agents for the amyloid fibrils. Various external factors like temperature, pH, pressure, ionic strength, the presence of metal ions and chemical molecules have been found to play important roles in the formation and disintegration of amyloid fibrils. Many antibodies, aromatic drugs, and polyphenolic compounds have been found to destroy preformed fibrils or inhibit formation of amyloid fibrils. However, the uses of these compounds are limited due to their cytotoxicity.[2] Thus, it is critically important to develop an efficient analytical tool that enables high-throughput screening for these diseases by decreasing the beta sheet content. Proteins, having large electric dipoles, respond to external electromagnetic radiation. It is anticipated that the application of electric field may have a role in reducing the content of β-sheets in the protein and is the major focus of this study. Few simulation studies, regarding the effect of electric field on protein conformation are found in the literature but those are performed at very high electric field strength which is not practically feasible.[3] A recent experimental study reveals that static DC electric field of small strength (8×106 V m-1) can effectively disintegrate human serum albumin (HSA) fibrils.[4] Fibrillation of human serum albumin, an amyloidogenic model protein due to its tendency to aggregate in vitro, has been studied extensively under different experimental conditions. However, HSA being a natively α-helical protein does not form fibrils under normal circumstances. The HSA fibrils are prepared incubating the protein in presence of 50% (v/v) ethanol in 20 mM Tris-HCl buffer of pH 7.0 at 37°C for 24 h. Herein, we have studied the effect of the application of an AC electric field on preformed fibrils of HSA wherein the effect of oscillation frequencies are probed at constant voltage. For the purpose of application of electric field, an arrangement similar to electro wetting on dielectric (EWOD) setup is used. Oscillating electric fields of various frequencies (0-100 Hz) at a fixed voltage of 8×106 V m-1 are applied on HSA fibril droplets for a period of 5 minutes. The disintegration process has been monitored by Thioflavin T fluorescence and circular dichroism spectroscopy. From the experimental observations it has been found that at a frequency 20 Hz, maximum reduction in β-sheet content is observed. This suggests that at a frequency of 20 Hz, the fibrillar structure is significantly disrupted due to the perturbation of various non-covalent (hydrophobic and electrostatic) interactions, responsible for the stability of fibrils. The application of AC voltage induced dynamic morphology change results in the generation of induced stress inside the HSA fibril droplets, and may be responsible for the reduction of the β-sheet content. The study may prove to be a precursor to a new methodology in the field of therapeutic treatment of neurological disorders and providing better understandings of the underlying mechanism of protein misfolding.

References

[1]   Rauk, A. (2008). Dalton Trans., 10, 1273-1282.

[2]   Wersinger, C., Sidhu, A. (2003). Neurosci.  Lett., 342, 124–128.

[3]   Lugli, F., Toschi, F., Biscarini, F., Zerbetto F. (2010). J. Chem. Theory Comput., 6, 3516–3526.

[4]   Pandey, N. K., Mitra, S., Chakraborty, M., Ghosh, S., Sen, S., Dasgupta, S., DasGupta, S. (2014). J. Phy. D: Appl. Phys, 47, 305401.