effect on TDG RD conformation. An increase of RD resonances inhibitor order us was measured Inhibitors,Modulators,Libraries when adding increasing amounts of SUMO 1 over TDG. We were also able to detect a gradual decrease of signal intensities for some resonances of the TDG C terminus in presence of SUMO 1 which indicates a modifica tion of the C terminal dynamics and conformation upon SUMO 1 intermolecular binding to SBMs. Remarkably, the non covalent interaction of SUMO 1 and the cova lent SUMO 1 modification of TDG induce a perturba tion of the same TDG C terminal resonances. This effect is obviously more pronounced for SUMO 1 conju gation than for the non covalent binding and leads to the only consistent interpretation that cis and trans SUMO 1 target at least one identical region of TDG CAT, the C terminal SUMO binding motif.

To confirm this interaction, we have acquired a 15N 1H HSQC spectrum on 15N labeled SUMO 1 in presence of TDG. Despite we observed some slight signal perturbations upon TDG addition it seems rather Inhibitors,Modulators,Libraries to be induced by weak, non specific inter actions. However, an overall 2 fold decrease of SUMO 1 signal intensity in the presence of TDG was noticed with exception of its N terminal resi dues that remain unchanged. Hence, the SUMO 1 population bound to TDG cannot be detected on the 15N 1H HSQC spectrum of 15N labeled SUMO 1 as already observed for SUMO 1 conjugated to TDG. Only the remaining free SUMO 1 molecules are detected. Taken together, our data indicate that non covalent interac tions between SUMO 1 and TDG exist, but do not directly involve the TDG N terminus which is in accor dance with previous studies.

Inhibitors,Modulators,Libraries SUMO 1 does not interact with TDG E310Q Having observed the importance of at least the C terminal SBM also in the case of covalent sumoylation of TDG, we decided to further analyze the SUMO 1 interaction sites within TDG CAT. Since two SUMO binding motifs had been previously proposed, one at the amino and another at the carboxy terminal part of TDG CAT, we wanted to determine which SBM mediates the N and or C terminal conformational changes which we were able to detect by NMR. We have produced three SBM mutants by either Inhibitors,Modulators,Libraries mutating the SBM1 or SBM2 or both similarly to Mohan and co workers. The 15 N labeled proteins were initially analyzed by NMR and circular dichroism spectroscopy.

Our data show that the D133A mutation of the conserved DIVII SUMO recognition sequence of the amino terminal SBM leads to a signifi cant misfolding of the protein and consequent aggrega tion and thus cannot be considered for further interaction studies with SUMO 1. Such a misfolding could be assigned to the experimental conditions or heterologous protein overexpression Dacomitinib in E. coli but it is not observed, however, for wild type TDG or the TDG E310Q mutant that are produced and investigated under the same conditions. It should also be noticed that the IVII motif, with exception of the D133 residue, is not solvent accessible in both the non and SUMO modified TDG CAT structures. While the www.selleckchem.com/products/PD-0332991.html D133A