The tumbling dynamics of a 20-mer HIV-1 RNA stem loop 3 spin-labeled at the 5' position were probed in the nanosecond time range. This RNA interacted with the HIV-1 nucleocapsid Zn-finger protein, 1-55 NCp7, and specialized stopped-flow EPR revealed concomitant kinetics of probe immobilization from milliseconds to seconds. RNA stem loop 3 is highly conserved in HIV, while NCp7 is critical to HIV-RNA packaging and annealing. The 5' probe did not perturb RNA melting or the NCp7/RNA interaction monitored by gel shift and fluorescence. The 5'-labeled RNA tumbled with a subnanosecond isotropic correlation time (approximately 0.60 ns at room temperature) reflecting both local viscosity-independent bond rotation of the probe and viscosity-dependent diffusion of 40-60% of the RNA. The binding of NCp7 to spin-labeled RNA stem loop 3 in a 1:1 ratio increased the spin-labeled tumbling time by about 40%. At low ionic strength with a ratio of NCp7 to RNA >or=3 (i.e., an NCp7 to nucleotide ratio
TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin label was attached at the N-terminal position to interrogate the dynamics of the HIV-1 nucleocapsid Zn-finger protein, NCp7. NCp7 is a 6.4 kDa 55-mer critical to the recognition, packaging, and efficient reverse transcription of viral RNA that has stem-loop structures, such as the RNA Stem-loop 3 used in this work. The NCp7, made by solid-phase peptide synthesis with TOAC incorporated into the α-carbon backbone at the N-terminal “0” position, showed analytical purity and biological activity. EPR spectra of the N-terminal TOAC indicated rapid temperature-sensitive motion of the probe (≤ 0.33 ns correlation time) on the flexible N-terminal segment. This N-terminal TOAC-NCp7 reported a RNA-NCp7 interaction at a 1:1 ratio of NCp7 to RNA which caused the tumbling time to be slowed from about 0.3 ns to about 0.5 ns. NCp7 is a largely disordered protein that adapts to its RNA targets. However, as shown by circular dichroism, ≥ 90% TFE (trifluoroethanol, an α-helix enhancer) caused the TOAC-NCp7 without zinc in its fingers to change to a fully helical conformation, while the TOAC spin label was concurrently reporting a tumbling time of well over a nanosecond as the N-terminal TOAC became inflexibly enfolded. Even with TFE present, the existence of intact Zn-finger regions in NCp7 prevented complete formation of helical structure, as shown by circular dichroism, and decreased the N-terminal TOAC tumbling time, as shown by EPR. This study demonstrated TOAC at the N-terminal of NCp7 to be a reporter for the considerable conformational lability of NCp7.
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