title: Mapping transition states of protein unfolding by protein engineering of ligand-binding sites.
abstract: We describe a method for probing the integrity and relative orientation of structural elements that are indirectly linked by ligands in protein complexes during protein folding. The effect of 3'-GMP on the rate constants of unfolding of wild-type barnase and several mutants has been studied. By comparing the rates of unfolding of wild-type and mutant proteins, we show that the interaction between His102 and 3'-GMP is fully retained in the transition state compared with the folded state, while the interaction between Glu60 and the ligand is partly retained and that of Lys27 is broken. Our data suggest that the transition state has a partly formed ligand binding site in which the guanine binding loop containing Glu60 and the loop containing His102 are formed at the sides of the beta-sheet but the docking of the N terminus of the second alpha-helix containing Lys27 on the beta-sheet is disrupted. The active site of barnase in complexes is thus partly retained in the transition state of unfolding. Although the ligand could in principle perturb the unfolding pathway, there is independent evidence that indicates that similar structural changes occur upon unfolding of unligated barnase.
authors.affiliation: M.R.C. Unit for Protein Function and Design, Cambridge University Chemical Laboratories, U.K.
authors.forename: J
authors.initials: J
authors.lastname: Sancho
authors.forename: E M
authors.initials: EM
authors.lastname: Meiering
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D001426:Bacterial Proteins; D001665:Binding Sites; D004926:Escherichia coli; D006157:Guanosine Monophosphate / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry*; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D006157:Guanosine Monophosphate; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease