Surface accessibility of hew lysozyme

INTRODUCTION: Nowadays structural biology represents an advanced frontier for a detailed understanding of life processes. Several projects of structural genomics are presented world-wide, since predictable or experimentally derived structures, mostly of proteins, represent a solid basis for engineering new therapeutic or diagnostic tools. However, some missing details still exist as the biological functions result to be not simply correlated to protein shapes and flexibility. The presence of “hot spots” on the molecular surface, always including the protein active sites (1,2), and the anomalous activity of some non-competitive enzyme inhibitors (3) suggest that around the protein surface a non-uniform motion of solvent and other molecules occurs. NMR seems to be perfectly suited to investigate on this new dimension of structural biology, since two techniques, i.e. water-protein Overhauser (4) effects and paramagnetic perturbation profiles (5), independently or in a combined way can be used to probe the accessibility of the protein surface towards molecules with different polarity. The results obtained from both NMR methods for a series of small proteins such as BPTI, tendamistat and single chain monellin (5,6) suggest that only a limited number of water molecules reside at the protein surface for a time long enough to yield sizeable intermolecular NOEs. The active sites of these proteins never have such tightly bound water molecules nearby. Accordingly, TEMPOL, a stable uncharged nitroxide, efficiently approaches the latter protein moieties due to the reduced hindrance from solvent molecules. Furthermore, there are protein regions where bound water molecules, organised in strong hydration sites, prevent the access of the nitroxide. MATERIALS AND METHODS: 13C-1H HSQC, 1D and 2D ePHOGSY spectra of 5 mM HEW lysozyme were acquired at 308 K and pH 3.8, using a Bruker Avance 600 spectrometer equipped with a Silicon Graphics Indy workstation. Paramagnetic attenuations were induced by the presence of 50 mM TEMPOL (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl). The enzyme inhibitor (NAG)3 was 7.5 mM to ensure full complexation of lysozyme. RESULTS: Paramagnetic attenuation profiles and water-protein nuclear Overhaser effects observed for HEW lysozyme, obtained in the presence and in the absence of the competitive inhibitor (NAG)3, were analysed in terms of accessibility of water and TEMPOL molecules towards the protein surface. The combined use of ePHOGSY spectroscopy and TEMPOL induced perturbations resulted to be very powerful also to find, as in the previously reported case for BPTI (5), the internal water molecules.