NIH Research Festival
Background: Despite major advancements in development of antiretroviral therapy, currently there is no effective treatment to block the impact of HIV-Tat protein which is released extracellularly and can cause activation of lymphocytes, glial cells and neurotoxicity. The structure of extracellular Tat remains unknown. Methods: Recombinant Tat protein was produced in E.coli, purified to >95% purity and stored frozen at -80 C until used. Atomic force microscopy (AFM) and circular dichroism were used to study the structure of HIV-Tat protein under near-physiological conditions. Results: About 8% of Tat population was in monomeric state, the rest being in aggregated form. The aggregates ranged in size from dimers, trimers, tetramers to large oligomers (50-mers and larger). Both monomeric and mulitmeric forms of Tat presented a globular shape. The reducing agent dithiothreitol broke down the large aggregates, leading to a population of smaller aggregates, but structures smaller then the monomer were found as well, indicating that the molecule is prone to rupture under reducing conditions. Circular dichroism measurements suggested that 20% of the sample’s structure is alpha-helical, which likely represents the aggregated state, since monomers were few. Cu2SO4 induced dimerization and larger aggregates of HIV-Tat. Conclusion: HIV-Tat protein easily forms large aggregates that have partial alpha helical structure and globular shape. The aggregation is increased by divalent cations and decreased by reducing agents. Future studies: Further studies involving protein concentration, salt increase, temperature variation and time of storage in liquid state will be done to characterize the aggregated state and its reversibility.
Scientific Focus Area: Biomedical Engineering and Biophysics
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