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J. Biol. Chem., Vol. 281, Issue 13, 8347-8356, March 31, 2006
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From the Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
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.
Received for publication, October 3, 2005 , and in revised form, January 11, 2006.
* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the University of Manitoba; it was initiated with funding from the Medical Research Council of Canada and the Manitoba Health Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1S–5S and Table 1S.
1 To whom correspondence should be addressed. Tel.: 204-474-6697; Fax: 204-474-7608; E-mail: joneil{at}cc.umanitoba.ca.
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