title: The Dynamic Landscape of the Full-Length HIV-1 Transactivator of Transcription.
abstract: The type 1 human immunodeficiency virus (HIV-1) transactivator of transcription (Tat) is a small RNA-binding protein essential for viral gene expression and replication. It has also been shown to bind to a large number of human proteins and to modulate many different cellular activities. We have used nuclear magnetic resonance (NMR) spectroscopy and hydrogen exchange chemistry to measure backbone dynamics over the millisecond to picosecond time scales. Sequential backbone assignment was facilitated by several isotope labeling schemes, including uniform labeling, site-specific labeling, and unlabeling. (15)N NMR relaxation parameters were measured and analyzed by reduced spectral density mapping and the Lipari-Szabo Model-Free approach to characterize the backbone dynamics on the picosecond to nanosecond time scale. The results indicate that the protein exists in an extended disordered conformational ensemble. NMR relaxation dispersion profiles show that on the millisecond time scale no conformational exchange is detected for any of the residues, supporting the model of a disordered backbone. NMR chemical shift differences from random coil values suggest that some segments of the protein have a modest propensity to fold; comparison to X-ray diffraction structures of Tat complexes indicates that some segments of the protein function through an induced-fit mechanism whereas other segments likely operate by conformational selection. Surprisingly, measured hydrogen exchange rates are higher than predicted for a disordered polymer, but this is explained as being caused by the high net charge on the protein that enhances base-catalyzed hydrogen exchange. The dynamics results provide a deeper understanding of the protein conformational ensemble and form a foundation for future studies of the conformational changes that accompany the formation of the superelongation complex that activates viral transcription.
authors: Department of Chemistry, University of Manitoba , Winnipeg, Manitoba R3T 2N2, Canada.
authors: Vu
authors: V
authors: To
authors: Department of Chemistry, University of Manitoba , Winnipeg, Manitoba R3T 2N2, Canada.
authors: Edis
authors: E
authors: Dzananovic
authors: Department of Chemistry, University of Manitoba , Winnipeg, Manitoba R3T 2N2, Canada.
authors: Sean A
authors: SA
authors: McKenna
authors: Department of Chemistry, University of Manitoba , Winnipeg, Manitoba R3T 2N2, Canada.
authors: Joe
authors: J
authors: O'Neil
mesh_terms: D015497:HIV-1 / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000502:physiology*
chemical_list: D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus