Limited searching in the conformational space is one of the major obstacles for investigating protein dynamics by numerical approaches. For this reason, classical all-atom molecular dynamics (MD) simulations of proteins tend to be confined to local energy minima, particularly when the bulk solvent is treated explicitly. To overcome this problem, we have developed a novel replica exchange protocol that uses modified force-field parameters to treat interparticle nonbonded potentials within the protein and between protein and solvent atoms, leaving unperturbed those relative to solvent-solvent interactions. We have tested the new protocol on the 18-residue-long tip of the P domain of calreticulin in an explicit solvent. With only eight replicas, we have been able to considerably enhance the conformational space sampled during a 100 ns simulation, compared to as many parallel classical molecular dynamics simulations of the same length or to a single one lasting 450 ns. A direct comparison between the various simulations has been possible thanks to the implementation of the weighted histogram analysis method, by which conformations simulated with modified force-field parameters can be assigned different weights. Interatom, inter-residue distances in the structural ensembles obtained with our novel replica exchange approach and by classical MD simulations compare equally well with those derived from NMR data. Rare events, such as unfolding and refolding, occur with reasonable statistical frequency. Visiting of conformations characterized by very small Boltzmann weights is also possible. Despite their low probability, such regions of the conformational space may play an important role in the search for local potential-energy minima and in dynamically controlled functions.

We present an interdisciplinary approach that, by incorporating a range of experimental and computational techniques, allows the identification and characterization of functional/immunogenic domains. This approach has been applied to ArtJ, an arginine-binding protein whose orthologs in Chlamydiae trachomatis (CT ArtJ) and pneumoniae (CPn ArtJ) are shown to have different immunogenic properties despite a high sequence similarity (60% identity). We have solved the crystallographic structures of CT ArtJ and CPn ArtJ, which are found to display a type II transporter fold organized in two ␣-␤ domains with the arginine-binding region at their interface. Although ArtJ is considered to belong to the periplasm, we found that both domains contain regions exposed on the bacterial surface. Moreover, we show that recombinant ArtJ binds to epithelial cells in vitro, suggesting a role for ArtJ in host-cell adhesion during Chlamydia infection. Experimental epitope mapping and computational analysis of physicochemical determinants of antibody recognition revealed that immunogenic epitopes reside mainly in the terminal (D1) domain of both CPn and CT ArtJ, whereas the surface properties of the respective binding-prone regions appear sufficiently different to assume divergent immunogenic behavior. Neutralization assays revealed that sera raised against CPn ArtJ D1 partially reduce both CPn and CT infectivity in vitro, suggesting that functional antibodies directed against this domain may potentially impair chlamydial infectivity. These findings suggest that the approach presented here, combining functional and structure-based analyses of evolutionary-related antigens can be a valuable tool for the identification of crossspecies immunogenic epitopes for vaccine development.

The possibility of estimating equilibrium free-energy profiles from multiple non-equilibrium simulations using the fluctuation-dissipation theory or the relation proposed by Jarzynski has attracted much attention. Although the Jarzynski estimator has poor convergence properties for simulations far from equilibrium, corrections have been derived for cases in which the work is Gaussian distributed. Here, we examine the utility of corrections proposed by Gore and collaborators using a simple dissipative system as a test case. The system consists of a single methane-like particle in explicit water. The Jarzynski equality is used to estimate the change in free energy associated with pulling the methane particle a distance of 3.9 nm at rates ranging from ~0.1 to 100 m s(-1). It is shown that although the corrections proposed by Gore and collaborators have excellent numerical performance, the profiles still converge slowly. Even when the corrections are applied in an ideal case where the work distribution is necessarily Gaussian, performing simulations under quasi-equilibrium conditions is still most efficient. Furthermore, it is shown that even for a single methane molecule in water, pulling rates as low as 1 m s(-1) can be problematic. The implications of this finding for studies in which small molecules or even large biomolecules are pulled through inhomogeneous environments at similar pulling rates are discussed.

ABSTRACTEscherichia coliβ-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version ofEscherichia coliβ-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation.