Computational Evolution Protocol for Peptide Design.

Computational peptide design is useful for therapeutics, diagnostics, and vaccine development. To select the most promising peptide candidates, the key is describing accurately the peptide-target interactions at the molecular level. We here review a computational peptide design protocol whose key feature is the use of all-atom explicit solvent molecular dynamics for describing the different peptide-target complexes explored during the optimization. We describe the milestones behind the development of this protocol, which is now implemented in an open-source code called PARCE. We provide a basic tutorial to run the code for an antibody fragment design example. Finally, we describe three additional applications of the method to design peptides for different targets, illustrating the broad scope of the proposed approach.

Toward a unified scoring function for native state discrimination and drug-binding pocket recognition.

Protein folding and receptor-ligand recognition are fundamental processes for any living organism. Although folding and ligand recognition are based on the same chemistry, the existing empirical scoring functions target just one problem: predicting the correct fold or the correct binding pose. We here introduce a statistical potential which considers moieties as fundamental units. The scoring function is able to deal with both folding and ligand pocket recognition problems with a performance comparable to the scoring functions specifically tailored for one of the two tasks. We foresee that the capability of the new scoring function to tackle both problems in a unified framework will be a key to deal with the induced fit phenomena, in which a target protein changes significantly its conformation upon binding. Moreover, the new scoring function might be useful in docking protocols towards intrinsically disordered proteins, whose flexibility cannot be handled with the available docking software.

Peptide biosensors for anticancer drugs: Design in silico to work in denaturizing environment.

One of the main targets in current clinical oncology is the development of a cheap device capable of monitoring in real-time the concentration of a drug in the blood of a patient. This would allow fine-tuning the dosage according to the patient's metabolism, a key condition to reduce side effects. By using surface plasmon resonance and fluorescence spectroscopy we here show that short peptides designed in silico by a recently developed algorithm are capable of binding the anticancer drug irinotecan (CPT-11) with micromolar affinity. Importantly, the recognition takes place in the denaturating solution used in standard therapeutic drug monitoring to detach the drug from the proteins that are present in human plasma, and some of the peptides are capable of distinguishing CPT-11 from its metabolite SN-38. These results suggest that the in silico design of small artificial peptides is now a viable route for designing sensing units, opening a wide range of applications in diagnostic and clinical areas.

Thermal compaction of the intrinsically disordered protein tau: entropic, structural, and hydrophobic factors.

Globular denatured proteins have structural properties similar to those of random coils. Experiments on denatured proteins have shown that when the temperature is increased thermal compaction may take place, resulting in a reduction of their radius of gyration Rg to range between 5% and 35% of its initial value. This phenomenon has been attributed to various causes, namely entropic, hydrophobic, and structural factors. The intrinsically disordered protein tau, which helps in nucleating and stabilizing microtubules in the axons of the neurons, also undergoes a relevant compaction process: when its temperature is increased from 293 K to 333 K its gyration radius decreases by 18%. We have performed an atomistic simulation of this molecule, at the lowest and highest temperatures of the mentioned interval, using both standard molecular dynamics and metadynamics, in parallel with small-angle X-ray scattering experiments. Using the fit of the experimental data and a genetic algorithm to select the most probable configurations among those produced in both atomistic simulations (standard MD and metadynamics), we were able to compute relevant changes, related to the temperature increase, in the average angles between residues, in the transient secondary structures, in the solvent accessible surface area, and in the number of intramolecular H-bonds. The analysis of the data showed how to decompose the compaction phenomenon into three contributions. An estimate of the entropic contribution to the compaction was obtained using the changes in the mean values of the angles between contiguous residues. The computation of the solvent accessible surface at the two temperatures allowed an estimation of the second factor contributing to the compaction, namely the increase in the hydrophobic interaction. We also measured the change in the average number of residues temporarily being in α-helices, 3-helices, PP II helices, ß-sheets and ß-turns. Those changes in the secondary structure population produce a reduction in the contour length of the protein, yielding a structural contribution to the reduction of Rg. This analysis shows that in tau the entropic factor accounts for about 60% of the compaction, the hydrophobic factor for about 25%, and the change in the secondary structure for about 15%.

Self-assembly of mesogenic bent-core DNA nanoduplexes.

Short cylinder-like DNA duplexes, comprising 6 to 20 base pairs, self-assemble into semi-flexible chains, due to coaxial stacking interactions between their blunt ends. The mutual alignment of these chains gives rise to macroscopically orientationally ordered liquid crystal phases. Interestingly, experiments show that the isotropic-nematic phase boundary is sequence-dependent. We perform all-atom simulations of several sequences to gain insights into the structural properties of the duplex and correlate the resulting geometric properties with the observed location of the isotropic-nematic phase boundary. We identify in the duplex bending the key parameter for explaining the sequence dependence, suggesting that DNA duplexes can be assimilated to bent-core mesogens. We also develop a coarse-grained model for the different DNA duplexes to evaluate in detail how bending affects the persistence length and excluded volume of the aggregates. This information is fed into a recently developed formalism to predict the isotropic-nematic phase boundary for bent-core mesogens. The theoretical results agree with the experimental observations.

Gastric foreign body as a risk factor for gastric dilatation and volvulus in dogs.

OBJECTIVE: To evaluate whether the presence of a gastric foreign body (gFB) is a significant risk factor for gastric dilatation and volvulus (GDV) in dogs and to quantify the change in likelihood of developing GDV associated with the presence of a gFB. DESIGN: Retrospective case-control study. ANIMALS: 118 large- or giant-breed dogs treated surgically for an episode of GDV and 342 large- or giant-breed dogs (> 12 months old) that underwent abdominal surgery for reasons other than GDV. PROCEDURES: During exploratory celiotomy, all dogs underwent palpation and visual examination of the entire gastrointestinal tract. A foreign body was defined as nondigestible or slowly digestible material palpated during gastrointestinal tract examination that was causing clinical signs or was > 10 cm in length or > 2 cm in width. RESULTS: The incidence of gFBs was significantly higher in the group of dogs with GDV. The presence of a gFB, age, weight, and purebred status were significant risk factors for GDV. Odds ratios were calculated for gFB (OR, 4.920), age (OR, 1.157), weight (OR, 0.958) and purebred status (OR, 4.836). CONCLUSIONS AND CLINICAL RELEVANCE: Gastric foreign body was found to be a significant risk factor for GDV in dogs. The study findings suggested that a large- or giant-breed dog with a gFB was approximately 5 times as likely to develop GDV as a similar dog with no gFB. Results indicated that there was a strong correlation between gFB and GDV in dogs. However, further cohort studies are needed to determine whether there is a causal relationship between the presence of a gFB and the development of GDV in dogs.

Continuous thermal collapse of the intrinsically disordered protein tau is driven by its entropic flexible domain.

The tau protein belongs to the category of Intrinsically Disordered Proteins (IDP), which in their native state lack a folded structure and fluctuate between many conformations. In its physiological state, tau helps nucleating and stabilizing the microtubules' (MTs) surfaces in the axons of the neurons. Tau is mainly composed by two domains: (i) the binding domain that tightly bounds the MT surfaces and (ii) the projection domain that exerts a long-range entropic repulsive force and thus provides the proper spacing between adjacent MTs. Tau is also involved in the genesis and in the development of the Alzheimer disease when it detaches from MT surfaces and aggregates in paired helical filaments. Unfortunately, the molecular mechanisms behind these phenomena are still unclear. Temperature variation, rarely considered in biological studies, is here used to provide structural information on tau correlated to its role as an entropic spacer between adjacent MTs surfaces. In this paper, by means of small-angle X-ray scattering and molecular dynamics simulation, we demonstrate that tau undergoes a counterintuitive collapse phenomenon with increasing temperature. A detailed analysis of our results, performed by the Ensemble Optimization Method, shows that the thermal collapse is coupled to the occurrence of a transient long-range contact between a region encompassing the end of the proline-rich domain P2 and the first part of the repeats domain, and the region of the N-terminal domain entailing residues 80-150. Interestingly these two regions involved in the tau temperature collapse belong to the flexible projection domain that acts as an entropic bristle and regulates the MTs' architecture. Our results show that temperature is an important parameter that influences the dynamics of the tau projection domain, and hence its entropic behavior.

Temperature and solvent dependence of the dynamical landscape of tau protein conformations.

We report the variation with temperature of the ensemble distribution of conformations spanned by the tau protein in its dynamical states measured by small-angle X-ray scattering (SAXS) using synchrotron radiation. The SAXS data show a clear temperature variation of the distribution of occupied protein conformations from 293 to 318 K. More conformations with a smaller radius of gyration are occupied at higher temperature. The protein-solvent interactions are shown by computer simulation to be essential for controlling the dynamics of protein conformations, providing evidence for the key role of water solvent in the protein dynamics, as proposed by Giorgio Careri.

Defining the thrombotic risk in patients with myeloproliferative neoplasms.

Polycythemia vera (PV) and essential thrombocythemia (ET) are two Philadelphia-negative myeloproliferative neoplasms (MPN) associated with an acquired mutation in the JAK2 tyrosine kinase gene. There is a rare incidence of progression to myelofibrosis and myeloid metaplasia in both disorders, which may or may not precede transformation to acute myeloid leukemia, but thrombosis is the main cause of morbidity and mortality. The pathophysiology of thrombosis in patients with MPN is complex. Traditionally, abnormalities of platelet number and function have been claimed as the main players, but increased dynamic interactions between platelets, leukocytes, and the endothelium do probably represent a fundamental interplay in generating a thrombophilic state. In addition, endothelial dysfunction, a well-known risk factor for vascular disease, may play a role in the thrombotic risk of patients with PV and ET. The identification of plasma markers translating the hemostatic imbalance in patients with PV and ET would be extremely helpful in order to define the subgroup of patients with a significant clinical risk of thrombosis.

Ordered and chaotic dynamics of collective variables in a butane molecule.

We have simulated the dynamics of a butane molecule and computed the time evolution of two sets of collective variables: (a) internal variables (stretchings, bendings, and dihedral angle) and (b) variables derived from a principal component analysis (PCA). We have characterized each collective variable by a coherence time, the time needed to develop its chaotic behavior. The coherence times diminish significantly when the temperature is raised into and above the range where conformational transitions of the dihedral angle set in. Below this transition region the coherence times of some variables reach hundreds of picoseconds (principal components) or even nanoseconds (internal variables); moreover, there are large differences among variables, as their coherence time can be much larger or much smaller than the Lyapunov time of the whole molecule. This result reflects the prediction of Nekhoroshev's theorem. Crossing the transition region, the coherence times of both sets of variables drop to few picoseconds, and the differences among variables diminish. Still, the coordinates and velocities characterized by the largest fluctuations in the PCA appear to be also the most coherent ones, below and above the transition region.