Two new techniques, chemical cross-linking and X-Ray footprinting, are promising in generating data related to the 3D structures of proteins. In chemical cross-linking, reagents are used to generate covalent bonds between the side chains of specific residues; the distances constraints obtained are used to model the structure of the protein and to reveal the interaction regions in the case of protein complexes. In footprinting, irradiation of the sample with ionizing radiation generates hydroxyl radicals that react with the exposed side chains. By comparing the rates of oxidation of the isolated and complexed proteins, it is possible to determine which amino acids are involved in the protein/protein interaction sites. In the present study, Tif34p and Tif35p, proteins related to the translation initiation factors in eukaryotes were analyzed by these two methodologies. Tif34p belongs to the WD-repeat family, which are characterized by the formation of a B-propeller structure and contains a repetition region that is supposed to participate in the interaction between proteins.
Cross-linking experiments were carried out using commercially available DSS reagent and two home-made reagents. These reactions were carried out with protein/reagent ratio of 1:20 for 15 minutes. The footprinting experiments were carried out in the DFX beamline in the Brazilian Synchrotron Light Source Laboratory. Samples were irradiated for 1-5 seconds. In both cases, samples were transferred to ammonium bicarbonate buffer, digested with sequencing grade trypsin and analyzed via LC-MS/MS in a Q-Tof Ultima API mass spectrometer coupled to a capillary liquid chromatography system.
The use of cross-linkers with different lengths can afford more complete data set for structural modeling and in this study three different cross-linkers were used, with arm lengths of 11.4, 10,2 and 7,7 A. LC-MS analyses of the unreacted and reacted complexes showed several differential ions, which are probably related to the cross-linking reaction. MS/MS experiments are being performed in order to identify these potential candidates. Both the complex Tif34p/HisTif35p and Tif34p alone were also subjected to X-Ray footprinting experiments. Rates of oxidation of different peptides were calculated using data from LC-MS analysis. MS/MS was used to identify the specific sites of oxidation.