2 STRUCTURAL SUBDOMAINS OF BARSTAR DETECTED BY RAPID MIXING NMR MEASUREMENT OF AMIDE HYDROGEN-EXCHANGE

Equilibrium amide hydrogen exchange studies of barstar have been carri ed out at pH 6.7, 32 degrees C using one-and two-dimensional nuclear m agnetic resonance, An unusually large fraction of the backbone amide h ydrogens of barstar exchange too fast to be measured, and the exchange rates of only fifteen slow-exchanging amide sites including indole am ides of two tryptophans could be measured in the presence of 0 to 1.8 M guanidine hydrochloride (GdnHCl). Measurement of exchange occurring in tens of seconds in the unfolding transition region was possible by the use of a fast stopped-flow mixing method, The observed exchange ra tes have been simulated in the EX2 limit according to a two-process mo del that incorporates two exchange-competent states: a transiently unf olded state (U) in which many amide hydrogens are completely accessib le to solvent-exchange, and a near-native locally unfolded state (N), in which only one or a few amide hydrogens are completely accessible to solvent-exchange. The two-process model appears to account for the observed exchange behavior over the entire range of GdnHCl concentrati ons studied, For several measurable slow-exchanging amide hydrogens, t he free energies of production of exchange-competent states from the e xchange-incompetent native state are significantly higher than the fre e-energy of production of the equilibrium unfolded state from the nati ve state, when the latter is determined from circular dichroism-or flu orescence-monitored equilibrium unfolding curves, The result implies t hat U, which forms transiently in the strongly native-like conditions used for the hydrogen exchange studies, is higher in energy than the equilibrium-unfolded state. The higher energy of this transiently unfo lded exchange-competent state can be attributed to either proline isom erization or to the presence of residual structure, On the basis of th e free energies of production of exchange-competent states, the measur ed amide sites of barstar appear to define two structural subdomains-a three-helix unit and a two-beta-strand unit in the core of the protei n. (C) 1998 Wiley-Liss, Inc.