Protein surface accessibility probed by solvent and paramagnetic molecules

Proteins very unlikely interact randomly with their molecular environment. Water, the most ubiquitous and abundant molecule of life, certainly plays a major role in controlling the intermolecular dialog of proteins. Thus, understanding how proteins drive other molecules to specific surface regions to trigger the biological function should imply a preliminary accurate delineation of protein hydration. Afterwards, the way a given protein surface region becomes a binding site, through particular surface shape, amino acid composition and topology, could be investigated by probing the protein surface accessibility to molecules different from water. It is apparent that delineating the mechanisms of protein surface accessibility would be precious to expand the rational basis for designing modified enzymes with modulated activity or protein ligands with biotechnological relevance. A good wealth of information on protein accessibility has been offered by nuclear magnetic resonance, NMR, through dynamic studies of proteins involved in exchanging and binding processes. Studies on the effects induced by neutral paramagnetic probes on protein NMR signals have been proved to be of particular relevance in delineating the complex dynamics occurring at the protein-solvent interface. The fact that paramagnetic probes might be involved in biased approaches towards a protein surface, due to their affinity with specific amino acid side chains or structural determinants, has been considered and several EPR studies have confirmed that preferential interactions between TEMPOL and proteins hardly occur. Furthermore, for the typical neutral or anionic Gd(III) chelates currently used as MRI contrast agents no evidence has been found by relaxometric techniques of weak interaction with specific molecular sites, even in the crowded molecular environments typical of biological fluids. The way surface patches of several protein systems are accessible to TEMPOL, GdDTPA-BMA and Gd2L7, a newly designed probe, is here reviewed. The fact that, irrespective of size and chemical nature of the three used paramagnets, a common path for their approach to the protein surface can be observed, is of primary importance. This finding suggested that uneven mobility of solvent molecules near to the protein surface could determine preferential access to protein regions where water molecule diffusion is particularly fast. A combined analysis of data obtained from Molecular Dynamics simulations and ePHOGSY spectra seems to confirm complementarity between hydrated surface sites and paramagnet accessible regions of all the proteins which we have investigated so far.