Nitrotyrosine chelation of nuclear magnetic resonance shift probes in proteins: application to bovine pancreatic trypsin inhibitor.
The interactions of Pr(III) and Eu(III) with specifically nitrated derivatives of the basic bovine pancreatic trypsin inhibitor have been studied using optical spectroscopy and nuclear magnetic resonance (NMR) at 250 and 270 MHz. Stability constants for proton and metal binding to nitrotyrosines 10 and 21 determined optically are in good agreement with those from NMR. Observations of the Eu(III)-induced NMR shifts of the ring protons of nitrotyrosine 21 allowed calibration of the magnetic interactions for this binding site. The Pr(III)-induced shifts for several resolved nonexchangeable backbone proton resonances were compared with calculated shifts using the known x-ray structure. With several simplifying assumptions, the Pr(III)-induced shifts were used to assign one alpha-CH and five NH protons to compatible sets of backbone positions which are consistent with the known pH dependence and resistance to exchange with solvent D2O. Some of the more general aspects of lanthanide-induced shifts are discussed with reference to their use in proteins. Due to the complexities of the analysis of the shift data, the most straightforward use of this technique is in conjunction with the relaxation probe Gd(III) for measurement of intramolecular distances.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014359:Trypsin Inhibitor, Kazal Pancreatic; D014361:Trypsin Inhibitors; D014443:Tyrosine / Q000031:analogs & derivatives*
D014361:Trypsin Inhibitors; D014443:Tyrosine; D014359:Trypsin Inhibitor, Kazal Pancreatic