2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) 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 ef.cient 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. Electron Paramagnetic Resonance (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% trifluoroethanol [(TFE), 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.
- Spin label
- ψ RNA