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• W1968649149 abstract "Genome sequencing projects have provided us with coding information about thousands of new proteins; however, many of them have unknown functions. Structural proteomics aims to determine their three-dimensional (3D) structures, thus leading to hypotheses about their functions. The sequence of TT1725 from Thermus thermophilus HB8 is conserved in three predicted prokaryotic proteins with unknown functions, including Deinococcus radiodurans, Stigmatella aurantiaca, and Mycobacterium leprae [Fig. 1(A)]. Here, we have determined the crystal structure of the hypothetical protein, TT1725, at 1.7 Å resolution by multiwavelength anomalous dispersion (MAD) phasing from a selenium-containing protein crystal [Fig. 1(B)]. The selenomethionine-substituted protein was synthesized by an Escherichia coli cell-free protein synthesis system.1 (A) Sequence alignment of TT1725 with three prokaryotic proteins: D. radiodurans, S. aurantiaca, and M. leprae. Identical and similar residues are highlighted in black and gray, respectively. Secondary structure regions observed experimentally in this study are indicated (h for helix, b for β sheet) above the sequence. (B) Ribbon cartoon representation of TT1725. The ribbon diagram was generated with MOLSCRIPT15 and RASTER3D.16 (C) Conserved residues of TT1725 mapped on the molecular surface. Front view of TT1725 is depicted as a colored molecular surface; red indicates conserved identical residues; orange, semi-invariant residues; and white, nonconserved residues. The molecular surface was created by the program GRASP.17 (D) Electrostatic surface representation of TT1725, with blue and red surfaces representing positive and negative potential, respectively. The molecular surface was created by the program GRASP.17 Cloning and purification procedures have been described elsewhere for other T. thermophilus proteins.2 The SeMet TT1725 was synthesized by a cell-free protein synthesis system.1 Crystals were obtained at 20°C by the hanging-drop vapor-diffusion method, from a solution containing 1.9 mg/mL protein, 10 mM Tris, pH8.5, 2.5% polyethylene glycol 3350 (PEG3350), and 75 mM sodium sulfate. The plate-shaped crystals grew in 1 day to approximate dimensions of 0.2 × 0.2 × 0.02 mm. We carried out a three-wavelength MAD experiment using a charged-coupled area detector (Mar CCD) installed on the beamline BL44B2 at the SPring-8 synchrotron facility, Harima, Japan. MAD and native data were processed and scaled with the HKL2000 suite of programs.3 Data collection statistics are presented in Table I. The Matthew's coefficient, VM was determined as 2.83 Å3Da−1, resulting in a solvent content of 56.5%, with a single molecule in each asymmetric unit. SOLVE4 was used to locate the selenium sites and to calculate the phases, and RESOLVE4 was used to modify the density. The automatic-tracing procedure in ARP/wARP5 was used to build a main-chain model for 94 of the 102 amino acid residues. The rest of the molecule was built into the electron density map using O,6 after an inspection of both 2Fo-Fc and Fo-Fc maps, followed by a least-squares refinement toward the data collected by the native crystal using CNS7 with bulk solvent correction. This yielded the final structure, including 98 amino acids of TT1725 and 129 solvent water molecules. Refinement statistics are shown in Table I. The structure of TT1725 is shown in Figure 1(B). The overall fold of this protein consists of two α-helices on one side of the molecule, and a four-stranded antiparallel β-sheet on the other. The secondary structure elements are as follows: β1, residues 2–13; α1, residues 19–36; β2, residues 40–45; β3, residues 52–61; α2, residues 64–80; and β4, residues 85–97. There are many conserved residues in the α1 region of TT1725 [Fig. 1(A, C)]. In addition, there are many positively-charged residues in α1 [Arg17, Lys20, Lys22, Arg23, Lys27, Arg32, Lys34, Arg36; Fig. 1(D)]. Therefore, we suggest that this region binds to a protein with a negatively charged region or to nucleic acids. In an initial effort to gain clues about the biological function of TT1725, its structure was compared with the previously determined structures in the Protein Data Bank (PDB), using Dali.8 Ten structural homologs of TT1725 were identified (Dali Z-scores ranging from 8.2 to 6.3). Six of the 10 proteins, which are higher in rank, belong to the ferredoxin-like fold,9 which contains βαββαβ. This fold is found in a number of functionally unrelated and nonhomologous proteins, for example, carboxypeptidase G210 (PDB code: 1CG2 chain A, Z = 8.2), phenylalanyl-tRNA synthetase11 (PDB code: 1PYS chain A, Z = 8.0), Atx1 metallochaperone protein12 (PDB code: 1CC8 chain A, Z = 7.7), peptidase T13 (PDB code: 1FNO chain A, Z = 7.4), and formylmethanofuran14 (PDB code: 1FTR chain A, Z = 6.8). Four of these structures are displayed with TT1725 in Figure 2. The electrostatic potentials of these proteins are not similar to that of TT1725. Ribbon cartoon representations of TT1725 and four similar structures, identified by Dali.8 The color codes used are navy (β sheet) and red (α-helix) for the parts sharing similar folds with TT1725. The ribbon cartoons were generated with MOLSCRIPT15 and RASTER3D.16 (A) TT1725. (B) Carboxypeptidase G210 (PDB code: 1CG2). (C) Phenylalanyl-tRNA synthetase11 (PDB code: 1PYS) (D) Atx1 metallochaperone protein12 (PDB: 1CC8). (E) formylmethanofuran14 (PDB code: 1FTR). We thank H. Yamaguchi for his technical assistance in data collection at the SPring-8, and H. Hamana and Y. Kamewari for synthesizing the selenomethionine substituted TT1725 by cell-free synthesis. Protein Data Bank coordinates entry code: 1 J27." @default.
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• W1968649149 date "2003-10-14" @default.
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• W1968649149 title "Crystal structure of a hypothetical protein, TT1725, from<i>Thermus thermophilus</i>HB8 at 1.7 Å resolution" @default.
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• W1968649149 doi "https://doi.org/10.1002/prot.10412" @default.
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