Abstract: The homology modelling of the structures of the 162 type II modules from the giant multi-domain protein titin is reported. The package Modeller was used and implemented in an automated fashion using four experimentally solved structures as templates. Validation of the models was addressed in terms of divergence from the template and consensus of the alignments. The homology within the whole family of type II modules as well as with the templates is relatively high (30-40% identity and ca. 60% similarity). Comparison between the models of domains for which an NMR structure has been solved with the experimental solution gives an estimate of the quality. Our results allow us to distinguish a number or structurally relevant residues that are therefore conserved in the whole family and buried in the hydrophobic core from those residues that are exposed and conserved. These will be funtionally relevant. Comparison of exposed conserved patches for modules in different regions of the titin molecule suggests potential interaction surfaces. Our results may be tested directly for those modules whose binding partner is known.

Abstract: Protein structure determination by NMR spectroscopy has become an essential tool in biological laboratories and so new methods are necessary, to perform 3D-structure determinations automatically. In view of the larger number of sequences becoming available, for example, from genome projects, the structural determination on a large scale is required, which could not be carried out without a high degree of automation. One of the most time-consuming steps in protein structure determination is the spectral assignment procedure. This involves two main phases: a) sequence specific resonance assignment of the nmr signals; b) assignment of the NOESY spectra with collection of a list of distance constraints: the crucial step. Advanced iterative approaches have been suggested to automate the assignment of NOESY spectra and 3D-structure calculation. We suggest an approach that simulates "molecular replacement methods" now currently in use in crystallography. A structure, obtained for instance from homology modelling or x-ray coordinates, may be used as template to automatically provide an initial guess. This structure can be then routinely refined against the experimental NMR data. We have applied this approach to determine the structure of Phl p II allergen, a protein of 96 residues with an immunoglobulin-like fold, of which both nmr and x-ray structures were available.

Abstract: Taste receptors have been studied less than those of other stimuli. However, the availability of many agonists and the practical relevance of sweeteners have stimulated indirect studies of the interaction of sweet agonists with their receptor and the development of general models of the sweet receptor active site. Most sweeteners are small molecular weight compounds but there are also sweet macromolecules, both synthetic and natural, i.e., sweet proteins. Do they interact with the same receptor of low molecular weight compounds? There are several sweet proteins: miraculin , monellin, thaumatin, curculin, mabinlin, pentadin and brazzein, but only three of them, i.e. thaumatin, monellin and brazzein, have been studied from a structural point of view. Multiple alignment of the sequences of sweet proteins shows no similarity. There is also no obvious similarity among the structures of thaumatin, monellin and brazzein. How can we identify the protein glucophores? We made the assumption that they are similar to those of low molecular weight compounds and that all sweet compounds interact with the same receptor. In fact, our model for the sweet receptor (Temussi et al., 1984, 1991) is consistent also with macromolecules since the active site is depicted as an open cavity with a flat bottom. When trying to explain the sweet taste of a protein it's natural to assume the existence of some kind of "sweet finger", i.e., a protruding structural element hosting one or more glucophores. We sought to identify sweet fingers in the three sweet proteins whose structure is known. Detailed structure comparison of all loops in the structures of thaumatin, monellin and brazzein by means of DALI shows that each protein hosts a likely sweet finger in which the spatial arrangement of three key residues (an aromatic a hydrogen bond donor and a hydrogen bond acceptor) is consistent with our model of the receptor active site.

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