Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR |
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Authors: | Honoka Okazaki Chie Kaneko Miyuki Hirahara Satoru Watanabe Naoya Tochio Takanori Kigawa Chiaki Nishimura |
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Institution: | 1. Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan;2. NMR Pipeline Methodology Team, RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan |
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Abstract: | N-terminal domain of HIV-1 p24 capsid protein is a globular fold composed of seven helices and two β-strands with a flexible structure including the α4–5 loop and both N- and C-terminal ends. However, the protein shows a high tendency (48%) for an intrinsically disordered structure based on the PONDR VL-XT prediction from the primary sequence. To assess the possibility of marginally stabilized structure under physiological conditions, the N-terminal domain of p24 was destabilized by the addition of an artificial flexible tag to either N- or C-terminal ends, and it was analyzed using T1, T2, hetero-nuclear NOE, and amide-proton exchange experiments. When the C-terminal tag (12 residues) was attached, the regions of the α3–4 loop and helix 6 as well as the α4–5 loop attained the flexible structures. Furthermore, in the protein containing the N-terminal tag (27 residues), helix 4 in addition to the above-mentioned area including α3–4 and α4–5 loops as well as helix 6 exhibited highly disordered structures. Thus, the long-range effects of the existence of tag sequence was observed in the stepwise manner of the appearance of disordered structures (step 1: α4–5 loop, step 2: α3–4 loop and helix 6, and step 3: helix 4). Furthermore, the disordered regions in tagged proteins were consistent with the PONDR VL-XT disordered prediction. The dynamic structure located in the middle part (α3–4 loop to helix 6) of the protein shown in this study may be related to the assembly of the viral particle. |
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Keywords: | NTD N-terminal domain CTD C-terminal domain CLEANEX-PM Phase-modulated clean chemical exchange p24N p24 NTD protein which has the additional N-terminal tag p24C p24 NTD protein which has the additional C-terminal tag p24cont p24 NTD protein with the linker sequence |
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