Mercury and selenium concentrations in lanugo of free-ranging California sea lions in the southern Gulf of California, Mexico.

Total mercury ([THg]) and selenium ([TSe]) concentrations were determined in California sea lion (Zalophus californianus) lanugo from the Gulf of California in 2021 and 2022. Relationships with sex, morphometrics, and year were evaluated. Following toxicological thresholds of concern for piscivorous mammals, most pups had a [THg] < 10 ppm, one pup (2021) had a [THg] > 20 ppm, no pups had a [THg] > 30 ppm. Females had significantly higher [TSe] than males; sex did not influence [THg]. [THg] and [TSe] in 2022 were significantly higher in the general population and male cohorts compared to 2021. Significant negative correlations were observed between [THg], [TSe], and morphometrics (2021). These results indicate that, compared to other pinniped species, regional California sea lions may have a decreased likelihood of experiencing Hg-related adverse health effects. Year-related changes in element concentrations suggest continued monitoring of this population to assess pinniped, environmental, and potentially, human health.

Indicators of oxidative stress in leukocytes isolated from bottlenose dolphins (Tursiops truncatus) in response to a proinflammatory challenge.

Few studies have analyzed the indicators of oxidative stress in marine mammals following exposure to lipopolysaccharides (LPS); sex and maturity-related differences have not been explored. The objective of this study was to compare the indicators of oxidative stress following exposure to LPS for 24 and 48 h in isolated Pacific bottlenose dolphin (Tursiops truncatus; N = 12) leukocytes in relation to sex and maturity stage, using spectrophotometry. Following 48 h under experimental conditions (10 µg LPS mL-1), the leukocytes from males (n = 5) produced significantly more superoxide radical (O2•-; F (1, 8) = 13.965, p = 0.006) and displayed significantly greater activities of catalase (CAT; F (1, 8) = 9.465, p = 0.015) and glutathione S-transferase (GST; p = 0.028) compared to the leukocytes from females (n = 7). Following 48 h under experimental conditions, maturity-stage did not significantly influence the indicators of oxidative stress. Mature bottlenose dolphins (n = 7) had a significantly higher average daily dietary intake compared to immature bottlenose dolphins (n = 5; F (1, 10) = 5.825, p = 0.036). These results suggest that sex-related strategies for coping with a proinflammatory challenge may be present within the leukocytes from bottlenose dolphins, while potential maturity stage-related strategies require further investigation.

NADPH oxidase 2 inhibitors CPP11G and CPP11H attenuate endothelial cell inflammation & vessel dysfunction and restore mouse hind-limb flow.

First described as essential to the phagocytic activity of leukocytes, Nox2-derived ROS have emerged as mediators of a range of cellular and tissue responses across species from salubrious to deleterious consequences. Knowledge of their role in inflammation is limited, however. We postulated that TNFα-induced endothelial reactive oxygen species (ROS) generation and pro-inflammatory signaling would be ameliorated by targeting Nox2. Herein, we in silico-modelled two first-in-class Nox2 inhibitors developed in our laboratory, explored their cellular mechanism of action and tested their efficacy in in vitro and mouse in vivo models of inflammation. Our data show that these inhibitors (CPP11G and CPP11H) disrupted canonical Nox2 organizing factor, p47phox, translocation to Nox2 in the plasma membrane; and abolished ROS production, markedly attenuated stress-responsive MAPK signaling and downstream AP-1 and NFκB nuclear translocation in human cells. Consequently, cell adhesion molecule expression and monocyte adherence were significantly inhibited by both inhibitors. In vivo, TNFα-induced ROS and inflammation were ameliorated by targeted Nox2 inhibition, which, in turn, improved hind-limb blood flow. These studies identify a proximal role for Nox2 in propagated inflammatory signaling and support therapeutic value of Nox2 inhibitors in inflammatory disease.

Optimization of minimum set of protein-DNA interactions: a quasi exact solution with minimum over-fitting.

MOTIVATION: A major limitation in modeling protein interactions is the difficulty of assessing the over-fitting of the training set. Recently, an experimentally based approach that integrates crystallographic information of C2H2 zinc finger-DNA complexes with binding data from 11 mutants, 7 from EGR finger I, was used to define an improved interaction code (no optimization). Here, we present a novel mixed integer programming (MIP)-based method that transforms this type of data into an optimized code, demonstrating both the advantages of the mathematical formulation to minimize over- and under-fitting and the robustness of the underlying physical parameters mapped by the code. RESULTS: Based on the structural models of feasible interaction networks for 35 mutants of EGR-DNA complexes, the MIP method minimizes the cumulative binding energy over all complexes for a general set of fundamental protein-DNA interactions. To guard against over-fitting, we use the scalability of the method to probe against the elimination of related interactions. From an initial set of 12 parameters (six hydrogen bonds, five desolvation penalties and a water factor), we proceed to eliminate five of them with only a marginal reduction of the correlation coefficient to 0.9983. Further reduction of parameters negatively impacts the performance of the code (under-fitting). Besides accurately predicting the change in binding affinity of validation sets, the code identifies possible context-dependent effects in the definition of the interaction networks. Yet, the approach of constraining predictions to within a pre-selected set of interactions limits the impact of these potential errors to related low-affinity complexes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Consensus alignment for reliable framework prediction in homology modeling.

MOTIVATION: Even the best sequence alignment methods frequently fail to correctly identify the framework regions for which backbones can be copied from the template into the target structure. Since the underprediction and, more significantly, the overprediction of these regions reduces the quality of the final model, it is of prime importance to attain as much as possible of the true structural alignment between target and template. RESULTS: We have developed an algorithm called Consensus that consistently provides a high quality alignment for comparative modeling. The method follows from a benchmark analysis of the 3D models generated by ten alignment techniques for a set of 79 homologous protein structure pairs. For 20-to-40% of the targets, these methods yield models with at least 6 A root mean square deviation (RMSD) from the native structure. We have selected the top five performing methods, and developed a consensus algorithm to generate an improved alignment. By building on the individual strength of each method, a set of criteria was implemented to remove the alignment segments that are likely to correspond to structurally dissimilar regions. The automated algorithm was validated on a different set of 48 protein pairs, resulting in 2.2 A average RMSD for the predicted models, and only four cases in which the RMSD exceeded 3 A. The average length of the alignments was about 75% of that found by standard structural superposition methods. The performance of Consensus was consistent from 2 to 32% target-template sequence identity, and hence it can be used for accurate prediction of framework regions in homology modeling.

Protein docking along smooth association pathways.

We propose a docking method that mimics the way proteins bind. The method accounts for the dominant driving forces at the different length scales of the protein binding process, allowing for an efficient selection of a downhill path on the evolving receptor-ligand-free energy landscape. Starting from encounter complexes with as much as 10 A rms deviation from the native conformation, the method locally samples the six dimensional space of rigid-body receptor-ligand structures subject to a van der Waals constraint. The sampling is initially biased only by the desolvation and electrostatic components of the free energy, which capture the partial affinity of unbound structures that are more than 4 A away from the native state. Below this threshold, improved discrimination is attained by adding an increasing fraction of the van der Waals energy to the force field. The method, with no free parameters, was tested in eight different sets of independently crystallized receptor-ligand structures consistently predicting bound conformations with the lowest free energies and appropriate stability gap around 2 A from the native complex. This multistage approach is consistent with the underlying kinetics and internal structure of the free energy funnel to the bound state. Implications for the nature of the protein binding pathways are also discussed.

Dynamical view of the positions of key side chains in protein-protein recognition.

When a complex is constructed from the separately determined rigid structures of a receptor and its ligand, some key side chains are usually in wrong positions. These distortions of the interface yield an apparent loss in affinity and would unfavorably affect the kinetics of association. It is generally assumed that the interacting proteins should drive the appropriate conformational changes, leading to their complementarity, but this hypothesis does not explain their fast association rates. However, nanosecond explicit solvent molecular dynamics simulations of misfolded surface side chains from the independently solved structures of barstar, bovine pancreatic trypsin inhibitor, and lysozyme show that even before any receptor-ligand interaction, key side chains frequently visit the rotamer conformations seen in the complex. We show that these simple structural motifs can reconcile most of the binding affinity required for a rapid and highly specific association process. Side chains amenable to induced fit are also identified. These results corroborate that solvent-side chain interactions play a critical role in the recognition process. Our findings are also supported by crystallographic data.

Predictions of gene family distributions in microbial genomes: evolution by gene duplication and modification.

A universal property of microbial genomes is the considerable fraction of genes that are homologous to other genes within the same genome. The process by which these homologues are generated is not well understood, but sequence analysis of 20 microbial genomes unveils a recurrent distribution of gene family sizes. We show that a simple evolutionary model based on random gene duplication and point mutations fully accounts for these distributions and permits predictions for the number of gene families in genomes not yet complete. Our findings are consistent with the notion that a genome evolves from a set of precursor genes to a mature size by gene duplications and increasing modifications.

Scoring docked conformations generated by rigid-body protein-protein docking.

Rigid-body methods, particularly Fourier correlation techniques, are very efficient for docking bound (co-crystallized) protein conformations using measures of surface complementarity as the target function. However, when docking unbound (separately crystallized) conformations, the method generally yields hundreds of false positive structures with good scores but high root mean square deviations (RMSDs). This paper describes a two-step scoring algorithm that can discriminate near-native conformations (with less than 5 A RMSD) from other structures. The first step includes two rigid-body filters that use the desolvation free energy and the electrostatic energy to select a manageable number of conformations for further processing, but are unable to eliminate all false positives. Complete discrimination is achieved in the second step that minimizes the molecular mechanics energy of the retained structures, and re-ranks them with a combined free-energy function which includes electrostatic, solvation, and van der Waals energy terms. After minimization, the improved fit in near-native complex conformations provides the free-energy gap required for discrimination. The algorithm has been developed and tested using docking decoys, i.e., docked conformations generated by Fourier correlation techniques. The decoy sets are available on the web for testing other discrimination procedures. Proteins 2000;40:525-537.

Continuum electrostatic analysis of preferred solvation sites around proteins in solution.

To understand water-protein interactions in solution, the electrostatic field is calculated by solving the Poisson-Boltzmann equation, and the free energy surface of water is mapped by translating and rotating an explicit water molecule around the protein. The calculation is applied to T4 lysozyme with data available on the conservation of solvent binding sites in 18 crystallographically independent molecules. The free energy maps around the ordered water sites provide information on the relationship between water positions in crystal structure and in solution. Results show that almost all conserved sites and the majority of nonconserved sites are within 1.3 A of local free energy minima. This finding is in sharp contrast to the behavior of randomly placed water molecules in the boundary layer, which, on the average, must travel more than 3 A to the nearest free energy minimum. Thus, the solvation sites are at least partially determined by protein-water interactions rather than by crystal packing alone. The characteristic water residence times, obtained from the free energies at the local minima, are in good agreement with nuclear magnetic resonance experiments. Only about half of the potential sites show up as ordered water in the 1.7 A resolution X-ray structure. Crystal packing interactions can stabilize weak or mobile potential sites (in fact, some ordered water positions are not close to free energy minima) or can prevent water from occupying certain sites. Apart from a few buried water molecules that are strong binders, the free energies are not very different for conserved and nonconserved sites. We show that conservation of a water site between two crystals occurs if the positions of protein atoms, primarily contributing to the free energy at the local minimum, do not substantially change from one structure to the other. This requirement can be correlated with the nature of the side chain contacting the water molecule in the site.

Kinetics of desolvation-mediated protein-protein binding.

The role of desolvation in protein binding kinetics is investigated using Brownian dynamics simulations in complexes in which the electrostatic interactions are relatively weak. We find that partial desolvation, modeled by a short-range atomic contact potential, is not only a major contributor to the binding free energy but also substantially increases the diffusion-limited rate for complexes in which long-range electrostatics is weak. This rate enhancement is mostly due to weakly specific pathways leading to a low free-energy attractor, i.e., a precursor state before docking. For alpha-chymotrypsin and human leukocyte elastase, both interacting with turkey ovomucoid third domain, we find that the forward rate constant associated with a collision within a solid angle phi around their corresponding attractor approaches 10(7) and 10(6) M(-1)s(-1), respectively, in the limit phi approximately 2 degrees. Because these estimates agree well with experiments, we conclude that the final bound conformation must be preceded by a small set of well-defined diffusion-accessible precursor states. The inclusion of the otherwise repulsive desolvation interaction also explains the lack of aggregation in proteins by restricting nonspecific association times to approximately 4 ns. Under the same reaction conditions but without short range forces, the association rate would be only approximately 10(3) M(-1)s(-1). Although desolvation increases these rates by three orders of magnitude, desolvation-mediated association is still at least 100-fold slower than the electrostatically assisted binding in complexes such as barnase and barstar.

Free energy landscapes of encounter complexes in protein-protein association.

We report the computer generation of a high-density map of the thermodynamic properties of the diffusion-accessible encounter conformations of four receptor-ligand protein pairs, and use it to study the electrostatic and desolvation components of the free energy of association. Encounter complex conformations are generated by sampling the translational/rotational space of the ligand around the receptor, both at 5-A and zero surface-to-surface separations. We find that partial desolvation is always an important effect, and it becomes dominant for complexes in which one of the reactants is neutral or weakly charged. The interaction provides a slowly varying attractive force over a small but significant region of the molecular surface. In complexes with no strong charge complementarity this region surrounds the binding site, and the orientation of the ligand in the encounter conformation with the lowest desolvation free energy is similar to the one observed in the fully formed complex. Complexes with strong opposite charges exhibit two types of behavior. In the first group, represented by barnase/barstar, electrostatics exerts strong orientational steering toward the binding site, and desolvation provides some added adhesion within the local region of low electrostatic energy. In the second group, represented by the complex of kallikrein and pancreatic trypsin inhibitor, the overall stability results from the rather nonspecific electrostatic attraction, whereas the affinity toward the binding region is determined by desolvation interactions.

Promotion and prevention choices between stability and change.

Two situations involving choice between stability and change were examined: task substitution, which deals with choosing between resuming an interrupted activity and doing a substitute activity, and endowment, which deals with choosing between a possessed object and an alternative object. Regulatory focus theory (E. T. Higgins, 1997, 1998) predicts that a promotion focus will be associated with openness to change, whereas a prevention focus will be associated with a preference for stability. Five studies confirmed this prediction with both situational induction of and chronic personality differences in regulatory focus. In Studies 1 and 2, individuals in a prevention focus were more inclined than individuals in a promotion focus to resume an interrupted task rather than do a substitute task. In Studies 3-5, individuals in a prevention focus, but not individuals in a promotion focus, exhibited a reluctance to exchange currently possessed objects (i.e., endowment) or previously possessed objects.

Denaturants can accelerate folding rates in a class of globular proteins.

We present a lattice Monte Carlo study to examine the effect of denaturants on the folding rates of simplified models of proteins. The two-dimensional model is made from a three-letter code mimicking the presence of hydrophobic, hydrophilic, and cysteine residues. We show that the rate of folding is maximum when the effective hydrophobic interaction epsilon H is approximately equal to the free energy gain epsilon S upon forming disulfide bonds. In the range 1 < or = epsilon H/ epsilon S < or = 3, multiple paths that connect several intermediates to the native state lead to fast folding. It is shown that at a fixed temperature and epsilon S the folding rate increases as epsilon H decreases. An approximate model is used to show that epsilon H should decrease as a function of the concentration of denaturants such as urea or guanidine hydrochloride. Our simulation results, in conjunction with this model, are used to show that increasing the concentration of denaturants can lead to an increase in folding rates. This occurs because denaturants can destabilize the intermediates without significantly altering the energy of the native conformation. Our findings are compared with experiments on the effects of denaturants on the refolding of bovine pancreatic trypsin inhibitor and ribonuclease T1. We also argue that the phenomenon of denaturant-enhanced folding of proteins should be general.

Modeling the role of disulfide bonds in protein folding: entropic barriers and pathways.

The role of disulfide bonds in directing protein folding is studied using lattice models. We find that the stability and the specificity of the disulfide bond interactions play quite different roles in the folding process: Under some conditions, the stability decreases the overall rate of folding; the specificity, however, by yielding a simpler connectivity of intermediates, always increases the rate of folding. This conclusion is intimately related to the selection mechanism entailed by entropic driving forces, such as the loop formation probability, and entropic barriers separating the native and the many native-like metastable states. The folding time is found to be a minimum for a certain range of the effective disulfide bond interaction. Examination of a model, which allows for the formation of disulfide bonded intermediates, suggests that folding proceeds via a three-stage multiple pathways kinetics. We show that there are pathways to the native state involving only native-like intermediates, as well as those that are mediated by nonnative intermediates. These findings are interpreted in terms of the appropriate energy landscape describing the barriers connecting low energy conformations. The consistency of our conclusions with several experimental studies is also discussed.

Theoretical predictions of folding pathways by using the proximity rule, with applications to bovine pancreatic trypsin inhibitor.

We propose a phenomenological theory that accounts for entropic effects due to loop formation to predict pathways in the kinetics of protein folding. The theory, the basis of which lies in multiple folding pathways and a three-stage kinetics, qualitatively reproduces most of the kinetic measurements in the refolding of bovine pancreatic trypsin inhibitor. The resulting pathways show that nonnative kinetic transients are involved in the productive routes leading to the formation of native intermediates. Our theory emphasizes the importance of the random origin of chain folding initiation structures in directing protein folding.

Kinetics and thermodynamics of folding in model proteins.

Monte Carlo simulations on a class of lattice models are used to probe the thermodynamics and kinetics of protein folding. We find two transition temperatures: one at T theta, when chains collapse from a coil to a compact phase, and the other at Tf (< T theta), when chains adopt a conformation corresponding to their native state. The kinetics are probed by several correlation functions and are interpreted in terms of the underlying energy landscape. The transition from the coil to the native state occurs in three distinct stages. The initial stage corresponds to a random collapse of the protein chain. At intermediate times tau c, during which much of the native structure is acquired, there are multiple pathways. For longer times tau r (>> tau c) the decay is exponential, suggestive of a late transition state. The folding time scale (approximately tau r) varies greatly depending on the model. Implications of our results for in vitro folding of proteins are discussed.