title: Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR.
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.
authors: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan.
authors: Honoka
authors: H
authors: Okazaki
authors: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan.
authors: Chie
authors: C
authors: Kaneko
authors: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan.
authors: Miyuki
authors: M
authors: Hirahara
authors: NMR Pipeline Methodology Team, RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan.
authors: Satoru
authors: S
authors: Watanabe
authors: NMR Pipeline Methodology Team, RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan.
authors: Naoya
authors: N
authors: Tochio
authors: NMR Pipeline Methodology Team, RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan.
authors: Takanori
authors: T
authors: Kigawa
authors: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan. Electronic address: cnishimura@thu.ac.jp.
authors: Chiaki
authors: C
authors: Nishimura
mesh_terms: D000595:Amino Acid Sequence; D016655:HIV Core Protein p24 / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D016655:HIV Core Protein p24; D011993:Recombinant Fusion Proteins; C514799:p24 protein, Human Immunodeficiency Virus Type 1
keywords: HIV-1 p24 capsid protein; Intrinsically disordered protein; NMR; Protein dynamics; Protein folding