HIGH-PRESSURE NMR-STUDY OF THE DISSOCIATION OF ARC REPRESSOR

Different denatured states of Are repressor were characterized by one-dimensional and two-dimensional NMR and by fluorescence spectroscopy. Increasing pressure promoted sequential changes in the structure of Arc repressor: from the native dimer through a predissociated state to a denaturated molten globule monomer. A compact state (molten globule) of Arc repressor was obtained in the dissociation of Arc repressor by pressure whereas high temperature and urea induced dissociation and unfolding to less structured conformations. The NMR spectra of the monomer under pressure (up to 5.0 kbar) are typical of a molten globule, and they are considerably different from those of the native dimer and the thermally or chemically denatured monomer. The substantial line broadening and overlap of many resonances in the NMR spectra at high pressures indicate that there is interconversion between a number of different conformations of the molten globule at an intermediate exchange rate. The two-dimensional NOE spectra show that the pressure-denatured monomer retains substantial secondary structure. The presence of NOEs in the beta-sheet region in the dissociated state suggests that the intersubunit beta-sheet (residues 6-14) in the native-dimer is replaced by an intramonomer beta-sheet. Changes in 2D NMR spectra prior to dissociation indicate the existence of a predissociated state that may represent an intermediate in the folding and subunit association pathway of Arc repressor.