Macro- and micro-stabilities of the kringle 4 domain from plasminogen. The effect of ligand binding

1H-NMR spectra of kringle 4 from human plasminogen have been recorded over wide pH* and temperature ranges, both in the presence and in the absence of p-benzylaminesulfonic acid (BASA). Several resonances exhibit chemical shift differences between kringle folded and unfolded forms which are sufficiently well resolved to allow for a determination of equilibrium Van't Hoff enthalpies and entropies for unfolding. The interaction with BASA shifts the kringle unfolding temperature from approximately 335 degrees K to approximately 343 degrees K. The pH* range of stability is also wider for the complex than for the free kringle: in the acidic range the pH* of half-unfolding, pHu*, is decreased from 2.8 for the unligated polypeptide to approximately 2.0 in the presence of BASA, while in the basic range pHu*, shifts from approximately 10.8 to 11.5. However, in contrast with what is observed at acidic pH*, unfolding at basic pH* leads to irreversible denaturation and exhibits a sharp, order-disorder transition both in the presence and in the absence of ligand. The structural stabilization conferred by the ligand is accompanied by a drastic reduction of the average rate of 1H-2H exchange in 2H2O under conditions that preclude a major cooperative unfolding. Thus, macro- and micro-stabilities of kringle domains appear to be highly correlated.