The S1 and S2 subunits of the spike glycoprotein of the coronavirus which is responsible for the severe acute respiratory syndrome (SARS) have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures. An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the genomic organisation similarity generally exhibited by coronaviruses. The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein. The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity.

The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure-function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis.Key words: Monellin; sweet proteins; taste; NMR structure; surface accessibility; hydration The use of low-calorie sweeteners is of paramount importance for people affected by diseases, such as diabetes, hyperlipemia, and caries, that are more or less directly linked to the assumption of sugar. The design of safe sweeteners relies upon an in-depth understanding of the SAR of sweet molecules and of their interaction with the receptor. Most sweeteners are small molecular weight compounds (Moncrieff 1967), but there are also sweet macromolecules, both synthetic (Zaffaroni 1975) and natural (i.e., sweet proteins [Morris 1976]). Considering the enormous difference in size among low-molecular-weight sweeteners and sweet proteins, and even among proteins, it has often been doubted that these two classes interact with the same receptor. However, both immunochemical studies and models of the receptor active site hint that all sweet molecules probably interact with the same receptor. ELISA tests showed crossreactivity among antibodies raised against monellin (94 residues) with thaumatin (210 residues) and aspartame (2 residues), and hinted that the sequence TyrA13-AspA16 of

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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 ...