After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded ←→ unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.

Summary: PMUT allows the fast and accurate prediction (~80% success rate in humans) of the pathological character of single point amino acidic mutations based on the use of neural networks. The program also allows the fast scanning of mutational hot spots, which are obtained by three procedures: (1) alanine scanning, (2) massive mutation and (3) genetically accessible mutations. A graphical interface for Protein Data Bank (PDB) structures, when available, and a database containing hot spot profiles for all non-redundant PDB structures are also accessible from the PMUT server. Availability: PMUT is freely accessible at http://mmb2.pcb.ub.es:8080/PMut/ Contact: [email protected] Supplementary information: http://mmb2.pcb.ub.es:8080/PMutWeb/methodology.html

SUMMARY: HTHquery is a web-based service to determine if a protein structure has a helix-turn-helix structural motif which could bind to DNA. It is based on a similarity with a set of structural templates, the accessibility of a putative structural motif and a positive electrostatic potential in the neighbourhood of the putative motif. A set of scores are computed, based on each template, using a linear predictor. From the training set used, the predictor has a true positive rate of 83.5% and a false positive rate of 0.8%. The emphasis for the website is on providing a straightforward interface which can be easily used by a bench-based scientist. AVAILABILITY: HTHquery is implemented using a set of Perl scripts and C program and can be accessed freely on the website http://www.ebi.ac.uk/thornton-srv/databases/HTHquery.