SOLUTION STRUCTURE OF REACTIVE-SITE HYDROLYZED TURKEY OVOMUCOID 3RD DOMAIN BY NUCLEAR-MAGNETIC-RESONANCE AND DISTANCE GEOMETRY METHODS

The solution structure of reactive-site hydrolyzed turkey ovomucoid th ird domain (OMTKY3) was determined by n.m.r. methods. A total of 655 distance constraints was applied in a distance geometry/simulated anne aling approach to calculate a family of structures consistent with the n.m.r. data. The input data included 24 torsion angle constraints, 14 hydrogen bonds, 611 constraints derived from two-dimensional nuclear Overhauser enhancement spectroscopy data, and three disulfide bridges. Stereospecific assignments were included for the hydrogens of 26 beta -methylene groups and for seven isopropyl methyl groups (46% chiral as signments). OMTKY3 in solution retains the global fold and overall se condary structure of the intact inhibitor (OMTKY3) but exhibits local structural differences at and adjacent to the clip site. In particular the hydrogen-bonding network observed at the reactive-site of the int act inhibitor is disrupted, and the position of Tyr20 is altered in th e modified inhibitor. No evidence was found for ion pairing between th e oppositely charged termini at the clip site. Surprisingly in light o f numerous changes indicating that OMTKY3: is less stable than OMTKY3 , rotation of the Tyr20 ring was found to be slow in OMTKY3 at 30 deg rees C. In OMTKY3, slow rotation of the Tyr31 ring was observed only a t temperatures below 15 degrees C. The n.m.r. structures of OMTKY3 ar e compared here with the similarly calculated structures of OMTKY3. Th is represents the first comparison of an intact and modified (reactive -site clipped) proteinase inhibitor under identical conditions. On com parison with published X-ray structures of modified avian ovomucoid th ird domains from two other species, the present structure of OMTKY3 i n solution was found to resemble that of the Japanese quail protein (O MJPQ3) more closely than that of the more closely homologous silver p heasant protein (OMSVP3).