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Abstract: The HIV-1 transactivator of transcription (Tat) is a protein essential for virus replication. Tat is an intrinsically disordered RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven Cys residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48–57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of 6Histidine-tagged and isotopically enriched (in and ) recombinant HIV-1 Tat1−72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the backbone NMR resonances. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced, monomeric, and unfolded in aqueous solution at pH 4. [Copyright &y& Elsevier]

Tat (transactivator of transcription) is a small RNA-binding protein that plays a central role in the regulation of human immunodeficiency virus type 1 replication and in approaches to treating latently infected cells. Its interactions with a wide variety of both intracellular and extracellular molecules is well documented. A molecular understanding of the multitude of Tat activities requires a determination of its structure and interactions with cellular and viral partners. To increase the dispersion of NMR signals and permit dynamics analysis by multinuclear NMR spectroscopy, we have prepared uniformly 15N- and 15N/13C-labeled Tat-(1-72) protein. The cysteine-rich protein is unambiguously reduced at pH 4.1, and NMR chemical shifts and coupling constants suggest that it exists in a random coil conformation. Line broadening and multiple peaks in the Cys-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analyzed by spectral density and Lipari-Szabo approaches, both confirming the lack of structure throughout the length of the molecule, The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of Tat protein to interact with a wide variety of proteins and nucleic acid and support the concept of a natively unfolded protein. [ABSTRACT FROM AUTHOR]