Nuclear magnetic resonance and dynamic characterization of the intrinsically disordered HIV-1 Tat protein
| dc.contributor.author | Shojania, Shaheen | |
| 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.contributor.supervisor | O'Neil, Joseph D. (Chemistry) | en |
| 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.degree.discipline | Chemistry | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| 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.description.note | October 2007 | en |
| dc.format.extent | 12524352 bytes | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | http://hdl.handle.net/1993/2814 | |
| dc.language.iso | eng | en_US |
| dc.rights | open access | en_US |
| 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 | doctoral thesis | en_US |