title: Solution structural studies and low-resolution model of the Schizosaccharomyces pombe sap1 protein.
abstract: Sap1 is a DNA-binding protein involved in controlling the mating type switch in fission yeast Schizosaccharomyces pombe. In the absence of any significant sequence similarity with any structurally known protein, a variety of biophysical techniques has been used to probe the solution low-resolution structure of the sap1 protein. First, sap1 is demonstrated to be an unusually elongated dimer in solution by measuring the translational diffusion coefficient with two independent techniques: dynamic light-scattering and ultracentrifugation. Second, sequence analysis revealed the existence of a long coiled-coil region, which is responsible for dimerization. The length of the predicted coiled-coil matches estimates drawn from the hydrodynamic experimental behaviour of the molecule. In addition, the same measurements done on a shorter construct with a coiled-coil region shortened by roughly one-half confirmed the localization of the long coiled-coil region. A crude T-shape model incorporating all these information was built. Third, small-angle X-ray scattering (SAXS) of the free molecule provided additional evidence for the model. In particular, the P(r) curve strikingly demonstrates the existence of long intramolecular distances. Using a novel 3D reconstruction algorithm, a low resolution 3D model of the protein has been independently constructed that matches the SAXS experimental data. It also fits the translation diffusion coefficients measurements and agrees with the first T-shaped model. This low-resolution model has clearly biologically relevant new functional implications, suggesting that sap1 is a bifunctional protein, with the two active sites being separated by as much as 120 A; a tetrapeptide repeated four times at the C terminus of the molecule is postulated to be of utmost functional importance.
authors.affiliation: Department of Applied Physics and SSRL, Stanford University, Stanford, CA, 94305, USA.
authors.forename: M
authors.initials: M
authors.lastname: Bada
authors.forename: D
authors.initials: D
authors.lastname: Walther
authors.forename: B
authors.initials: B
authors.lastname: Arcangioli
authors.forename: S
authors.initials: S
authors.lastname: Doniach
authors.forename: M
authors.initials: M
authors.lastname: Delarue
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004058:Diffusion; D019281:Dimerization; D008027:Light; D008958:Models, Molecular; D008970:Molecular Weight; D011485:Protein Binding; D014176:Protein Biosynthesis; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D012996:Solutions; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C085981:Sap1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D012996:Solutions; D004247:DNA