Nonkinetic modeling of the mechanical unfolding of multimodular proteins: theory and experiments.

We introduce and discuss a novel approach called back-calculation for analyzing force spectroscopy experiments on multimodular proteins. The relationship between the histograms of the unfolding forces for different peaks, corresponding to a different number of not-yet-unfolded protein modules, is exploited in such a manner that the sole distribution of the forces for one unfolding peak can be used to predict the unfolding forces for other peaks. The scheme is based on a bootstrap prediction method and does not rely on any specific kinetic model for multimodular unfolding. It is tested and validated in both theoretical/computational contexts (based on stochastic simulations) and atomic force microscopy experiments on (GB1)(8) multimodular protein constructs. The prediction accuracy is so high that the predicted average unfolding forces corresponding to each peak for the GB1 construct are within only 5 pN of the averaged directly-measured values. Experimental data are also used to illustrate how the limitations of standard kinetic models can be aptly circumvented by the proposed approach.

An integrated approach for bioaccumulation assessment in mussels: towards the development of Environmental Quality Standards for biota.

The possible use of chemical concentrations measured in mussels (Mytillus galloprovincialis) for compliance checking against Environmental Quality Standards (EQS) established for biota is analyzed with the help of an integrated model. The model consists of a 3D planktonic module that provides biomasses in the different compartments, i.e., phytoplankton, zooplankton and bacteria; a 3D fate module that provides the concentrations of contaminants in the water column and in the sediments; and a 3D bioaccumulation module that calculates internal concentrations in relevant biotic compartments. These modules feed a 0D growth and bioaccumulation module for mussels, based on the Dynamic Energy Budget (DEB) approach. The integrated model has been applied to study the bioaccumulation of persistent organic pollutants (POPs) in the Thau lagoon (France). The model correctly predicts the concentrations of polychlorinated biphenyls (PCBs) and polychlorinated dibenzodioxins and dibenzofurans (PCDD/Fs) in mussels as a function of the concentrations in the water column and in phytoplankton. It also sheds light on the origin of the complexity associated with the use of EQS for biota and their conversion to water column concentrations. The integrated model is potentially useful for regulatory purposes, for example in the context of the European Water Framework (WFD) and Marine Strategy Framework Directives (MSFD).

Assessment of ecological risk from bioaccumulation of PCDD/Fs and dioxin-like PCBs in a coastal lagoon.

The ecological risk posed by dioxin-like PCBs and PCDD/Fs congeners mixtures in five organisms representative of the food web of the lagoon of Venice (Italy) was estimated by applying the US-EPA [US-EPA. Guidelines for Ecological Risk Assessment. EPA/630/R-95/002F. Final Report. U.S. Environmental Protection Agency, Washington, DC; 1998.] procedure. Experimental concentrations of dioxin-like PCBs and PCDD/Fs in sediments, water, and organisms in six lagoon zones were used to define the spatial distribution of the pollutant homolog patterns. The Principal Component Analysis of homolog patterns in biota permitted to remark the difference between PCDD/Fs and dioxin-like PCBs bioaccumulation. Dioxin-like PCBs were found almost unaltered in all selected organisms, while PCDD/Fs homologs underwent an enrichment of low chlorinated furans in fish. The ecological risk for the dioxin-like PCBs and PCDD/Fs was estimated in the selected lagoon zones according to the Hazard Quotient approach by comparing the biota experimental concentrations in TEQ with an Internal No Effect Concentration of the 2,3,7,8-TCDD, estimated from both Ambient Quality Criteria and literature toxicity data. A high potential risk was found for benthic biota (i.e. clam and crab) and for mussel, while a negligible risk was estimated for fish. Moreover, the ecological risk resulted higher in the central part of the lagoon, and lower in the northern lagoon. Finally, the comparison between the TEQ concentrations of each homolog in biota and the estimated ecological risk highlighted that the risk was determined essentially by lower chlorinated PCDD/Fs (i.e. pentachloro and hexachloro PCDD/Fs), and by pentachloro-PCBs. These compounds should be regarded as homologs of concern and preferentially investigated in environmental monitoring of sediment, water, and biota.

Knotting of random ring polymers in confined spaces.

Stochastic simulations are used to characterize the knotting distributions of random ring polymers confined in spheres of various radii. The approach is based on the use of multiple Markov chains and reweighting techniques, combined with effective strategies for simplifying the geometrical complexity of ring conformations without altering their knot type. By these means we extend previous studies and characterize in detail how the probability to form a given prime or composite knot behaves in terms of the number of ring segments N and confining radius R. For 50 < or =N < or =450 we show that the probability of forming a composite knot rises significantly with the confinement, while the occurrence probability of prime knots are, in general, nonmonotonic functions of 1R. The dependence of other geometrical indicators, such as writhe and chirality, in terms of R and N is also characterized. It is found that the writhe distribution broadens as the confining sphere narrows.

Elucidation of the disulfide-folding pathway of hirudin by a topology-based approach.

A theoretical model for the folding of proteins containing disulfide bonds is introduced. The model exploits the knowledge of the native state to favor the progressive establishment of native interactions. At variance with traditional approaches based on native topology, not all native bonds are treated in the same way; in particular, a suitable energy term is introduced to account for the special strength of disulfide bonds, as well as their ability to undergo intramolecular reshuffling. The model thus possesses the minimal ingredients necessary to investigate the much debated issue of whether the refolding process occurs through partially structured intermediates with native or non-native disulfide bonds. This strategy is applied to a context of particular interest, the refolding process of hirudin, a thrombin-specific protease inhibitor, for which conflicting folding pathways have been proposed. We show that the only two parameters in the model (temperature and disulfide strength) can be tuned to reproduce well a set of experimental transitions between species with different number of formed disulfides. This model is then used to provide a characterization of the folding process and a detailed description of the species involved in the rate-limiting step of hirudin refolding.

Learning effective amino acid interactions through iterative stochastic techniques.

The prediction of the three-dimensional structures of the native states of proteins from the sequences of their amino acids is one of the most important challenges in molecular biology. An essential task for solving this problem within coarse-grained models is the deduction of effective interaction potentials between the amino acids. Over the years, several techniques have been developed to extract potentials that are able to discriminate satisfactorily between the native and nonnative folds of a preassigned protein sequence. In general, when these potentials are used in actual dynamical folding simulations, they lead to a drift of the native structure outside the quasinative basin. In this article, we present and validate an approach to overcome this difficulty. By exploiting several numerical and analytical tools, we set up a rigorous iterative scheme to extract potentials satisfying a prerequisite of any viable potential: the stabilization of proteins within their native basin (less than 3-4 A RMSD). The scheme is flexible and is demonstrated to be applicable to a variety of parameterizations of the energy function, and it provides in each case the optimal potentials.