Date of Award

1-1-2009

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Content Description

1 online resource (xvii, 153 pages) : illustrations (some color)

Dissertation/Thesis Chair

Charles P Scholes

Committee Members

Carla Theimer, Keith Earle, Li Niu, Alexander Shekhtman

Keywords

Chaperone activity, Double Electron-Electron Resonance (DEER), Electron paramagnetic resonance (EPR), human immunodeficiency virus type 1(HIV 1), nucleocapsid protein (NC), NCp7, Transactivation Response Element (TAR), HIV (Viruses), HIV antibodies

Subject Categories

Biochemistry

Abstract

The nucleocapsid (NC) protein NCp7 of the human immunodeficiency virus type 1(HIV 1) is a small basic protein with two zinc finger motifs. NCp7 has key roles in virus replication and structure, which rely on its interactions with nucleic acids. We investigated the interaction of NCp7 with Transactivation Response Element (TAR) DNA which is critical for minus strand transfer during the HIV 1 reverse transcription. The EPR spin probe nitroxide was used as the reporter. Binding of NCp7 to TARDNA caused TARDNA condensation, as inferred from the probe tumbling time which markedly increased to several nanoseconds at a 4:1 NCp7 to TARDNA ratio, signifying a NCp7/TARDNA complex with restricted motion. To further investigate NCp7-mediated annealing of TARDNA to the complementary sequence TARRNA, a process which is important for virus replication, Double Electron-Electron Resonance (DEER) was used to directly monitor conformational changes in the TARDNA structure upon NCp7 binding. We found that in the absence of the acceptor TARRNA, NCp7 had only a limited inhibitory effect on the hairpin structure destabilization. In contrast, when both TARDNA and TARRNA were present, NCp7 facilitated formation of the transfer duplex product, and the destabilization of hairpin structure greatly increased. A systematic study of the annealing process of TARDNA with TARRNA hairpins in the presence of NCp7 was performed. These measurements helped to explain why acceptor TARRNA is required for significant annealing of TARDNA by NCp7, and supported the hypothesis that NCp7-mediated annealing of nucleic acids is a concerted process wherein the unfolding step occurs in synchrony with hybridization of complementary TARDNA and TARRNA. Taken as a whole, these studies demonstrated that the spin labeling and DEER measurement provide atomic resolution information of structure and binding interactions.

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