Enhancing the solubility of intrinsically disordered HIV-1 Tat protein at physiological pH and structural investigation by NMR spectroscopy

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Krishnamurthy, Kiran
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Human Immunodeficiency Virus-1 (HIV-1) Transactivator of transcription (Tat) protein is a 101-residue intrinsically disordered protein, responsible for enhancing the transcription process and ultimately viral replication. To understand its mechanistic role in enhancing transcription and viral replication, a study of its structure and dynamics in the presence of its binding partners is required. However, the protein is soluble only under acidic conditions (pH 4) and it precipitates at pH 7, which precludes the determination of its structure, dynamics and interactions under physiological conditions. Hence, the primary objective of this research was to solubilize Tat-protein near pH 7 so that further studies may be carried out to discern the mechanistic role of the Tat protein in viral replication. Multiple approaches were employed to improve the solubility of the Tat protein at pH 7. A sequence-specific nickel-assisted cleavage (SNAC) approach that involves cleaving the polyhistidine-tagged Tat was employed to produce full-length Tat protein minus the purification tag, but this was unsuccessful owing to an unpredictable internal cleavage site. Poly-anionic RNA from Torula yeast was used as a solubilizing agent and it was found to increase the solubility of Tat but Nuclear Magnetic Resonance (NMR) spectra were only marginally improved. Tat was observed to be insoluble at pH 4−7 in the presence of TAR (TransActivation Response) RNA, one of the binding partners of Tat which is vital for the functioning of the protein. Moreover, the solubility of Tat was tested in a solution containing HIV-1 TAR RNA and Torula yeast RNA. Here too, the protein was soluble but no major improvement in the NMR spectra of Tat was observed. Tat protein tagged with a supercharged sequence at the N-terminal was genetically engineered and expressed to test the effect of a high net-charge on Tat’s solubility. The increased net charge did not improve Tat’s solubility. Genetic engineering was used to replace seven cysteine residues of iii the Tat protein with aspartic acid to study the role of cysteine residues in the aggregation of the protein at neutral pH. This approach produced highly soluble Tat at pH 7 with well-resolved NMR spectra leading to the realization of the important role that Cys oxidation plays in the solubility of the protein. The carboxy terminal domain of RNA polymerase II (RNAP II) undergoes liquid-liquid phase separation in the presence of a crowding agent. With the expectation that Tat might be soluble in a liquid-liquid phase separated medium at physiological pH, a RNAP II domain fusion protein was expressed, but poor yields of proteolyzed protein precluded Tat solubility studies. Several analytical techniques were employed to characterize the structure of Tat protein. Fluorescence studies were carried out to understand the structural changes taking place as the pH is elevated. Somewhat surprisingly, the fluorescence spectra indicated that the single Trp residue resides in a solvent-restricted region of intrinsically-disordered Tat. Infrared spectroscopy was used to study the secondary structure of the protein and to quantify the fractions of different secondary structures comprising the Tat protein in the range of pH 4−7. Multi-dimensional solution- and solid-state NMR spectroscopy were employed as the primary analytical tool in the structural analysis of Tat protein. In addition to conventional indirect-detection NMR methods, 15N-direct-detection NMR experiments were attempted to help monitor solubility, structure and dynamics.