We introduce Paircoil2, a new version of the Paircoil program, which uses pairwise residue probabilities to detect coiled-coil motifs in protein sequence data. Paircoil2 achieves 98% sensitivity and 97% specificity on known coiled coils in leave-family-out cross-validation. It also shows superior performance compared with published methods in tests on proteins of known structure.

Many virulence factors secreted from pathogenic Gram-negative bacteria are autotransporter proteins. The final step of autotransporter secretion is C 3 N-terminal threading of the passenger domain through the outer membrane (OM), mediated by a cotranslated C-terminal porin domain. The native structure is formed only after this final secretion step, which requires neither ATP nor a proton gradient. Sequence analysis reveals that, despite size, sequence, and functional diversity among autotransporter passenger domains, >97% are predicted to form parallel ␤-helices, indicating this structural topology may be important for secretion. We report the folding behavior of pertactin, an autotransporter passenger domain from Bordetella pertussis. The pertactin ␤-helix folds reversibly in isolation, but folding is much slower than expected based on size and native-state topology. Surprisingly, pertactin is not prone to aggregation during folding, even though folding is extremely slow. Interestingly, equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the C-terminal half of the protein. Examination of the pertactin crystal structure does not reveal any obvious reason for the enhanced stability of the C terminus. In vivo, slow folding would prevent premature folding of the passenger domain in the periplasm, before OM secretion. Moreover, the extra stability of the C-terminal rungs of the ␤-helix might serve as a template for the formation of native protein during OM secretion; hence, vectorial folding of the ␤-helix could contribute to the energyindependent translocation mechanism. Coupled with the sequence analysis, the results presented here suggest a general mechanism for autotransporter secretion.parallel ␤-sheet ͉ contact order ͉ outer membrane protein ͉ protein structure prediction ͉ virulence factor

The human eye lens is composed of layers of elongated fiber cells packed with crystallin proteins at concentrations up to 400 mg/ml. Human gD-crystallin (HgD-Crys), one of the three major g-crystallins, is a monomeric, two-domain protein found in the lens nucleus, the central region of the lens formed earliest during development. Genetic screens for mutations resulting in cataract in mice identified three mutations affecting mouse g-crystallins. These amino acid substitutions were introduced into HgD-Crys by site-specific mutation of the cloned gene. The three mutant proteins L5S, V75D, and I90F were expressed and purified from E. coli. Equilibrium unfolding/refolding experiments were performed to measure the thermodynamic stability of the mutant proteins compared to wild type. Wild-type HgD-Crys was previously shown to exhibit a three-state unfolding/refolding pathway. This pathway is sequential with the N-terminal domain unfolding first, followed by the C-terminal domain. L5S and V75D also displayed three-state unfolding/refolding transitions with populated intermediates. In both cases, the first transition midpoint was shifted to lower denaturant concentrations, 0.7 M GdnHCl for L5S and 0.8 M for V75D compared to 2.2 M for the wild type. I90F exhibited a two-state unfolding/refolding transition with a single midpoint at 1.7 M. The mutant proteins all exhibited decreased thermal stability compared with wild type. Kinetic unfolding experiments confirmed that wild type unfolded through a three-state mechanism. The N-terminal domains of L5S and V75D unfolded extremely fast (t 1/2 @2 s) at lower denaturant concentrations than those required for wild type. I90F was globally destabilized and unfolded through a two-state mechanism faster than wild type. These results support models of cataract formation in which generation of partially unfolded intermediates -whether due to mutation or to covalent damage -are precursors to the aggregated cataractous states responsible for light scattering.

The ability to predict structure from sequence is particularly important for toxins, virulence factors, allergens, cytokines, and other proteins of public health importance. Many such functions are represented in the parallel beta-helix and beta-trefoil families. A method using pairwise beta-strand interaction probabilities coupled with evolutionary information represented by sequence profiles is developed to tackle these problems for the beta-helix and beta-trefoil folds. The algorithm BetaWrapPro employs a "wrapping" component that may capture folding processes with an initiation stage followed by processive interaction of the sequence with the already-formed motifs. BetaWrapPro outperforms all previous motif recognition programs for these folds, recognizing the beta-helix with 100% sensitivity and 99.7% specificity and the beta-trefoil with 100% sensitivity and 92.5% specificity, in crossvalidation on a database of all nonredundant known positive and negative examples of these fold classes in the PDB. It additionally aligns 88% of residues for the beta-helices and 86% for the beta-trefoils accurately (within four residues of the exact position) to the structural template, which is then used with the side-chain packing program SCWRL to produce 3D structure predictions. One striking result has been the prediction of an unexpected parallel beta-helix structure for a pollen allergen, and its recent confirmation through solution of its structure. A Web server running BetaWrapPro is available and outputs putative PDB-style coordinates for sequences predicted to form the target folds.