This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein splicing is a post-translational event in which an internal protein segment, the intein, is excised from a precursor with concomitant ligation of the flanking sequences, the N- and the C- exteins. Protein splicing is a self catalysed reaction, involving four nucleophilic displacements. The first step of this reaction, the N-S acyl shift, is, on the face of it, a thermodinamically unfavoured process. The current hypothesis to explain why the first step of the splicing reaction occurs is that the scissile amide bond may be in an unusual conformation, either cis or even twisted-trans, and this might provide a thermodynamic leg-up for the reaction. Currently there is no information on the solution conformation of the scissile amide bond in the intein. One way to investigate the nature of this bond is by isotope edited NMR on a GyrA protein in which the C-terminal extein residue is labelled with 13C. This protein has been obtaind by expressed protein ligation, reacting a 15N labelled Gyra to a thioester peptide which has the same sequence as the N-terminal extein. Preliminary HNCO and HSQC NMR experiments how that the 1J N-C of the scissile amide bond is unusual. This suggest that there is no twisting in the amide bond , but the carbonyl seems to be involved in a strong hydrogen bond . Our current hypotesis is that the hydrogen bond might be responsible for a shift in the amid bond from the N-C=O to the N=C-OH form, which would be more susceptible to the nucleophilic attack from the Cys1 thiol. In order to test this hypothesis we identified some residues which are important for the splicing and we're planning to repeat the same NMR measurements as before on proteins in which these residues are mutated or modified. Monitoring the 1J N-C coupling constant in the new proteins we'll try to track the residue responsible for the hydrogen bond donation. One of the residue to modify is the Cys 1; this amino-acid is in fact primarly involved in the N-S acyl shift. Since this residue is fundamental for the ligation, it can be modified only after the GyrA protein has been ligated to the peptide. Cys1 can be transformed in a Met by methylation with methly iodide. Analytical experiments on the ligated protein indicate that it is possible to methylate three residues in the protein. Trypsin digestion and MALDI-TOF analysis on the digested crude revealed that the three Cys have selectively reacted. NMR measurements on the methylated GyrA will allow us to identify or exclude the Cys1 as hydrogen bond donator.
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