NMR studies of the dynamics of the multidomain protein urokinase-type plasminogen activator

u-PA (urokinase-type plasminogen activator or urokinase) has been studied under a variety of solution conditions by 1-D and 2-D NMR spectroscopy. Very high quality spectra could be obtained from the recombinant protein despite the high molecular mass (46 kDa) by appropriate choice of solution conditions; mildly acidic pH and low ionic strength were found to be optimal. Comparison of spectra of u-PA with spectra of the isolated kringle and protease domains, the EGF-kringle pair, and a synthetic peptide from the kringle-protease linker region, enabled sequential assignments in the u-PA spectrum to be made for kringle resonances, and domain-specific assignments for many others. Simulations of line shapes in both 1-D and 2-D spectra enabled effective correlation times for the different domains, both isolated and in the intact protein, to be determined. These have permitted a model of the u-PA dynamics to be put forward involving extensive, but not unrestricted, motion between the different domains.