AMIDE EXCHANGE-RATES IN ESCHERICHIA-COLI ACYL CARRIER PROTEIN - CORRELATION WITH PROTEIN-STRUCTURE AND DYNAMICS

The acyl carrier protein (ACP) of Escherichia coli is a 77-amino acid, highly negatively charged three-helix protein that plays a central ro le in fatty acid biosynthesis. Previous NMR studies have suggested the presence of multiple conformations and marginally stable secondary st ructural elements. The stability of these elements is now examined by monitoring amide exchange in apo-ACP using NMR-based methods. Because ACP exhibits many rapid exchange rates, application of traditional iso tope exchange methods is difficult. In one approach, heteronuclear cor relation experiments with pulsed field-gradient coherence selection ha ve reduced the time needed to collect two-dimensional H-1-N-15 correla tion spectra to the point where measurement of exchange of amide proto ns for deuterium on the timescale of minutes can be made. In another a pproach, water proton selective inversion-exchange experiments were pe rformed to estimate the exchange rates of protons exchanging on timesc ales of less than a second. Backbone amide protons in the region of he lix II were found to exchange significantly more rapidly than those in helices I and III, consistent with earlier structural models suggesti ng a dynamic disruption of the second helix. Highly protected amides o ccur on faces of the helices that may pack into a hydrophobic core pre sent in a partially disrupted state.