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Rotational dynamics of HIV‐1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis
Author(s) -
Zhang Zhiwen,
Xi Xiangmei,
Scholes Charles P.,
Karim Christine B.
Publication year - 2008
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21064
Subject(s) - chemistry , spin label , rna , crystallography , circular dichroism , stereochemistry , electron paramagnetic resonance , peptide , biophysics , nuclear magnetic resonance , biochemistry , physics , gene , biology , membrane
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 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. 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. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 1125–1135, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com