Determining binding sites in protein-nucleic acid complexes by cross-saturation

Cross-saturation experiments have been shown to give accurate information r egarding the interacting surfaces in protein-protein and protein-RNA comple xes. The rate of magnetization transfer depends on a number of factors incl uding geometry, spin topology, and effective correlation times. To assess t he influence of these variables on such experiments, and to determine the r ange of applicability of the technique, we have simulated the time-course o f magnetization transfer across the interface in a variety of protein-nucle ic acid complexes (434 Cro, SRY, MetJ and U1A), each having a different bin ding geometry. The simulations have been carried out primarily to provide i nformation about the experimentally accessible targets for selective satura tion, such as the anomeric protons and the imino protons of the nucleic aci d. Saturation of either of these groups of signals leads to partial excitat ion throughout the nucleic acid molecule, and the resulting transfer of sat uration to the labelled protein moiety can be readily detected by the reduc tion in intensity of particular peaks in the HSQC spectrum of the protein. The simulations show that information can be obtained about the residues in contact with the nucleic acid without any need for deuteration. Experiment al cross-saturation data have been obtained from the Mbp1-DNA complex and i nterpreted in conjunction with the simulations to map out the binding surfa ce in detail.