期刊
JOURNAL OF VIROLOGICAL METHODS
卷 164, 期 1-2, 页码 35-42出版社
ELSEVIER
DOI: 10.1016/j.jviromet.2009.11.021
关键词
HIV; Tat; Transcriptional regulator; Intrinsically disordered protein; Cysteine-rich; NMR spectroscopy
资金
- Natural Sciences and Engineering Research Council of Canada
- University of Manitoba
- Medical Research Council of Canada
- Manitoba Health Research Council
- Canadian Foundation for Innovation
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 6x Histidine-tagged and isotopically enriched (in N-15 and C-13) recombinant HIV-1 Tat(1-72) (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. (C) 2009 Elsevier B.V. All rights reserved.
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