Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity.Interactions of proteins with other molecules can ultimately be ascribed to their surface features. Direct studies of protein surface accessibility are emerging as a new dimension of structural studies of proteins, particularly because repeated observations in either solution (1, 2) or crystal state (3, 4) have pointed out that proteins have regions where small and uncharged organic molecules, even those different from their physiological ligands, preferentially approach the molecular surface and also account for allosteric disruption of substrate binding (5, 6). We have shown that these "hot spots" of the protein surface can be easily mapped by a surface survey based on paramagnetic perturbation of conventional NMR spectra (7,8).The surface properties are dictated by the relative position and specific features of exposed residues, but even detailed knowledge of the protein architecture may not be sufficient for a thorough description of surface properties because of the intrinsic disorder of these residue side chains. The complex properties of the protein surface are modulated by a variety of factors (e.g. electrostatics, hydrophobicity, and hydrogen bond ability) but share a common unifying feature: hydration. The blanket of water covering the protein surface is the actual interface between the solution environment and the underlying modulations. The possibility of exploiting the blanket resides mainly on two of its features, namely, the variable thickness of the water layers and the fact that residence times of water molecules vary from point to point (9). Since the pioneering studies on protein hydration by Wü thrich's and co-workers (10, 11), it has been well established that NMR is a reliable technique with which to detect water molecules bound to proteins (12). The early approaches were burdened by delicate hardware requirements, but now, thanks to developments in gradientcontrolled sequences (12-16), intermolecular nuclear Overhauser effects between water and protein molecules can be routinely measured and correlated to overall protein hydration.It has recently been proposed that the ability of surface ...