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 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.
Shojania
Shaheen
S
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
O'Neil
Joe D
JD
D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005612:Freeze Drying; D015696:Gene Products, tat / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D019810:Sodium Azide / Q000494:pharmacology; D012996:Solutions; D013696:Temperature; D054322:tat Gene Products, Human Immunodeficiency Virus
D004791:Enzyme Inhibitors; D015696:Gene Products, tat; D010446:Peptide Fragments; D012996:Solutions; D054322:tat Gene Products, Human Immunodeficiency Virus; D006639:Histidine; D019810:Sodium Azide; D003545:Cysteine