Se Bok Jang

Novel crystal form of the ColE1 rom protein

The RNA I modulator protein (Rom) acts as a co-regulator of ColE1 plasmid copy number by binding to RNA kissing hairpins and stabilizing their interaction. The structure of Rom has been determined in a new crystal form from X-ray diffraction data to 2.5 A resolution. In this structure, a dimer of the 57-amino-acid protein is found in the asymmetric unit. Each subunit consists almost entirely of two antiparallel alpha-helices joined by a short hairpin bend. The dimer contains a non-crystallographic twofold axis and forms a highly regular four-alpha-helical bundle. The structural packing in this novel crystal form is different from previously known Rom structures. The asymmetric unit contains one dimer, giving a crystal volume per protein weight (V(M)) of 1.83 A(3) Da(-1) and a low solvent content of 30%. Strong packing interactions and low solvation are characteristic of the structure. The Rom protein was cocrystallized with the Tar-Tar* kissing hairpin RNA. Although the electron-density maps do not show bound RNA, altered conformations in the side chains of Rom that are known to be involved in RNA binding have been identified. These results provide additional information about Rom protein conformational flexibility and suggest that the presence of a highly charged polymer such as RNA can promote tight packing of an RNA-binding protein, even when the RNA itself is not observed in the crystal.

Crystallization and preliminary X-ray analysis of tryptophan synthase α-subunits from Escherichia coli

Tryptophan synthase alpha-subunit (alphaTS) catalyzes the cleavage of indole-3-glycerolphosphate to glyceraldehyde-3-phosphate and indole, which is channelled to the active site of the associated beta-subunit (betaTS), where it reacts with serine to yield the amino acid tryptophan in tryptophan biosynthesis. The alphaTS from Escherichia coli is a 268 amino-acid protein with no disulfide bonds or prosthetic groups. Although the crystallization of the subunits from E. coli has been attempted over many years, there have been no reports of an X-ray structure. To explore the molecular origin of the conformational stabilization mechanism of alphaTS, the alpha-subunit protein was overexpressed in E. coli and crystallized using the hanging-drop vapour-diffusion method at 298 K. A native data set to 2.8 A resolution was obtained from a flash-cooled crystal upon exposure to Cu Kalpha X-rays. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 162.27, b = 44.48, c = 71.52 A, beta = 106.56 degrees. The asymmetric unit contains two molecules of alphaTS, giving a crystal volume per protein mass (V(M)) of 2.16 A(3) Da(-1) and a solvent content of 43.18%.

Biological and structural characterizations of mutations in X-linked spondyloepiphyseal dysplasia tarda

Spondyloepiphyseal dysplasia tarda (SEDT), an X-linked genetic disease manifesting itself in a disproportionate skeletal structure, is caused by mutations in the SEDL gene. Four missense mutations (S73L, V130D, F83S, and D47Y) have been identified by molecular diagnosis as disease-causing SEDT. Nevertheless, how SEDL mutations disrupt the skeletal structure and cause disease remains unknown. We report here the cloning, expression, and characterization of three different missense mutations (S73L, V130D, and D47Y) in mouse SEDL. The overexpression of the D47Y mutation was mainly observed in the supernatant but those of the S73L and V130D mutations are shown in the insoluble pellets. The substitution of the S73L mutation induces the exposure to hydrophobic amino acids and causes aggregation. That of V130D might break hydrophobic interaction and decrease the secondary structure. The CD spectra of three mutants (S73L, V130D, and D47Y) showed that the α-helices decreased more than that of wild-type SEDL. The F83S (stop) mutant might suggest a large conformational change as the mutant V130D. In order to visualize conformational changes in mutated structures, we used molecular modeling techniques minimizing the total energy. These results could provide the biological characterization and conformational information of the SEDL mutants and suggest the clinical severity of the disorder among human SEDL patients.