HIV-1 Tat is a natively unfolded protein - The solution conformation and dynamics of reduced HIV-1 Tat-(1-72) by NMR spectroscopy

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 N-15- and N-15/C-13- 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.