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dc.contributor.supervisor O'Neil, Joseph D. (Chemistry) en
dc.contributor.author Shojania, Shaheen
dc.date.accessioned 2007-09-14T15:21:52Z
dc.date.available 2007-09-14T15:21:52Z
dc.date.issued 2007-09-14T15:21:52Z
dc.identifier.uri http://hdl.handle.net/1993/2814
dc.description.abstract The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an 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 Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine 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 polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free 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 the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein. en
dc.format.extent 12524352 bytes
dc.format.mimetype application/pdf
dc.language.iso en_US
dc.rights info:eu-repo/semantics/openAccess
dc.subject HIV-1 Tat en
dc.subject NMR
dc.subject relaxation
dc.subject dynamics
dc.subject protein characterization
dc.subject intrinsic disorder
dc.title Nuclear magnetic resonance and dynamic characterization of the intrinsically disordered HIV-1 Tat protein en
dc.type info:eu-repo/semantics/doctoralThesis
dc.degree.discipline Chemistry en
dc.contributor.examiningcommittee Al-Hashimi, Hashim M. (Chemistry and Biophysics, University of Michigan) Peeling, Jim (Radiology, University of Manitoba) Hruska, Frank (Chemistry, University of Manitoba) en
dc.degree.level Doctor of Philosophy (Ph.D.) en
dc.description.note October 2007 en


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