title: Analytical ultracentrifugation studies of oligomerization and DNA-binding of TtCarH, a Thermus thermophilus coenzyme B12-based photosensory regulator.
abstract: Thermus thermophilus transcriptional factor TtCarH belongs to a newly discovered class of photoreceptors that use 5'-deoxyadenosylcobalamin (AdoB12) as the light-sensing chromophore. Photoregulation relies on the repressor activity of AdoB12-bound oligomers in the dark, which light counteracts by oligomer disruption due to AdoB12 photolysis. In this study, we investigated TtCarH self-association and binding to DNA in the dark and in the light using analytical ultracentrifugation (AUC) methods, both sedimentation velocity (SV) as well as equilibrium (SE). From a methodological point of view, this study shows that AUC can provide hydrodynamic insights in cases where light is a crucial determinant of solution properties. For the light-sensitive TtCarH, absorbance as well as interference AUC data yielded comparable results. Sedimentation coefficients and whole-body hydrodynamic analysis from SV experiments indicate that in solution apo-TtCarH and light-exposed AdoB12-TtCarH are predominantly aspherical, ellipsoidal monomers, in accord with SE data. By comparison, AdoB12-TtCarH exists as a more compact tetramer in the dark, with smaller forms such as dimers or monomers remaining undetected and low levels of larger oligomers appearing at higher protein concentrations. AUC analyses indicate that in the dark AdoB12-TtCarH associates as a tetramer with DNA but forms smaller complexes in the apo form or if exposed to light. The self-association and DNA-binding properties of TtCarH deduced from AUC are consistent with data from size-exclusion and DNA-binding gel-shift assays. AUC analyses together with hydrodynamic modeling provide insights into the AdoB12- and light-dependent self-association and DNA-binding of TtCarH.
authors.affiliation: Departamento de Química Física, Facultad de Química, Universidad de Murcia, 30071, Murcia, Spain.
authors.forename: Ana I
authors.initials: AI
authors.lastname: Díez
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: Ortiz-Guerrero
authors.forename: Alvaro
authors.initials: A
authors.lastname: Ortega
authors.forename: Montserrat
authors.initials: M
authors.lastname: Elías-Arnanz
authors.forename: S
authors.initials: S
authors.lastname: Padmanabhan
authors.forename: José
authors.initials: J
authors.lastname: García de la Torre
mesh_terms: D019540:Area Under Curve; D001426:Bacterial Proteins / Q000737:chemistry*; D002845:Chromatography / Q000379:methods; D003038:Cobamides / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D057446:Hydrodynamics; D008027:Light; D015233:Models, Statistical; D010777:Photochemistry / Q000379:methods; D020283:Photoreceptors, Microbial / Q000737:chemistry*; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000737:chemistry*; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D003038:Cobamides; D020283:Photoreceptors, Microbial; D004247:DNA; C000913:cobamamide