Free Energy, Enthalpy and Entropy from Implicit Solvent End-Point Simulations

Free energy is the key quantity to describe the thermodynamics of biological systems. In this perspective we consider the calculation of free energy, enthalpy and entropy from end-point molecular dynamics simulations. Since the enthalpy may be calculated as the ensemble average over equilibrated simulation snapshots the difficulties related to free energy calculation are ultimately related to the calculation of the entropy of the system and in particular of the solvent entropy. In the last two decades implicit solvent models have been used to circumvent the problem and to take into account solvent entropy implicitly in the solvation terms. More recently outstanding advancement in both implicit solvent models and in entropy calculations are making the goal of free energy estimation from end-point simulations more feasible than ever before. We review briefly the basic theory and discuss the advancements in light of practical applications. \ua9 2018 Fogolari, Corazza and Esposito

Protein Aggregation Measurement through Electrical Impedance Spectroscopy

The paper presents a novel methodology to measure the fibril formation in protein solutions. We designed a bench consisting of a sensor having interdigitated electrodes, a PDMS hermetic reservoir and an impedance meter automatically driven by calculator. The impedance data are interpolated with a lumped elements model and their change over time can provide information on the aggregation process. Encouraging results have been obtained by testing the methodology on K-casein, a protein of milk, with and without the addition of a drug inhibiting the aggregation. The amount of sample needed to perform this measurement is by far lower than the amount needed by fluorescence analysis

Occurrence and Etiology of Brown Apical Necrosis on Persian (English) Walnut Fruit.

In 1998, a severe fruit drop was observed in Italy, principally on cv. Lara Persian (English) walnut (Juglans regia). Dropped fruit showed a brown patch at the blossom end and blackening and rot of inner tissues. The disease, called brown apical necrosis (BAN), was investigated on fruit collected in Italy and France in 1999. In 2000, studies were carried out in three walnut orchards located in Italy and in France to substantiate the etiology of BAN. Isolations performed from inner diseased fruit tissues yielded several fungi, in decreasing frequency of isolation: species of Fusarium and Alternaria, and one species each of Cladosporium, Colletotrichum, and Phomopsis. However, only Fusarium spp. were recovered from stigmas of BAN-affected fruit. The fungi associated with BAN-diseased fruit and species composition differed among locations and over time, confirming results obtained in previous investigations. The species of Fusarium used in pathogenicity tests reproduced BAN-disease symptoms when inoculated on fruit, whereas an Alternaria alternata isolate caused only limited necrosis of the style. However, the role of the other fungi commonly isolated from BAN-diseased fruit remains to be defined. The walnut blight pathogen, Xanthomonas arboricola pv. juglandis, occasionally was isolated from BAN-diseased fruit. No correlation was found between the extent of external brown patches and the size of inner lesions. Repeated isolations from and inoculations of fruit demonstrated that BAN can be considered a complex disease, and the inner infections originate from the style of the fruit

Automation of peak-tracking analysis of stepwise perturbed NMR spectra

6noWe describe a new algorithmic approach able to automatically pick and track the NMR resonances of a large number of 2D NMR spectra acquired during a stepwise variation of a physical parameter. The method has been named Trace in Track (TinT), referring to the idea that a gaussian decomposition traces peaks within the tracks recognised through 3D mathematical morphology. It is capable of determining the evolution of the chemical shifts, intensity and linewidths of each tracked peak.The performances obtained in term of track reconstruction and correct assignment on realistic synthetic spectra were high above 90% when a noise level similar to that of experimental data were considered. TinT was applied successfully to several protein systems during a temperature ramp in isotope exchange experiments. A comparison with a state-of-the-art algorithm showed promising results for great numbers of spectra and low signal to noise ratios, when the graduality of the perturbation is appropriate. TinT can be applied to different kinds of high throughput chemical shift mapping experiments, with quasi-continuous variations, in which a quantitative automated recognition is crucial.openopenBanelli, Tommaso; Vuano, Marco; Fogolari, Federico; Fusiello, Andrea; Esposito, Gennaro; Corazza, AlessandraBanelli, Tommaso; Vuano, Marco; Fogolari, Federico; Fusiello, Andrea; Esposito, Gennaro; Corazza, Alessandr

Bluues server

Abstract Motivation: Electrostatic calculations are an important tool for deciphering many functional mechanisms in proteins. Generalized Born (GB) models offer a fast and convenient computational approximation over other implicit solvent-based electrostatic models. Here we present a novel GB-based web server, using the program Bluues, to calculate numerous electrostatic features including pKa-values and surface potentials. The output is organized allowing both experts and beginners to rapidly sift the data. A novel feature of the Bluues server is that it explicitly allows to find electrostatic differences between wild-type and mutant structures. Availability: The Bluues server, examples and extensive help files are available for non-commercial use at URL: http://protein.bio.unipd.it/bluues/. Contact: [email protected]

Scoring predictive models using a reduced representation of proteins: model and energy definition

BACKGROUND: Reduced representations of proteins have been playing a keyrole in the study of protein folding. Many such models are available, with different representation detail. Although the usefulness of many such models for structural bioinformatics applications has been demonstrated in recent years, there are few intermediate resolution models endowed with an energy model capable, for instance, of detecting native or native-like structures among decoy sets. The aim of the present work is to provide a discrete empirical potential for a reduced protein model termed here PC2CA, because it employs a PseudoCovalent structure with only 2 Centers of interactions per Amino acid, suitable for protein model quality assessment. RESULTS: All protein structures in the set top500H have been converted in reduced form. The distribution of pseudobonds, pseudoangle, pseudodihedrals and distances between centers of interactions have been converted into potentials of mean force. A suitable reference distribution has been defined for non-bonded interactions which takes into account excluded volume effects and protein finite size. The correlation between adjacent main chain pseudodihedrals has been converted in an additional energetic term which is able to account for cooperative effects in secondary structure elements. Local energy surface exploration is performed in order to increase the robustness of the energy function. CONCLUSION: The model and the energy definition proposed have been tested on all the multiple decoys' sets in the Decoys'R'us database. The energetic model is able to recognize, for almost all sets, native-like structures (RMSD less than 2.0 Å). These results and those obtained in the blind CASP7 quality assessment experiment suggest that the model compares well with scoring potentials with finer granularity and could be useful for fast exploration of conformational space. Parameters are available at the url:

Molecular models for intrastrand DNA G-quadruplexes

<p>Abstract</p> <p>Background</p> <p>Independent surveys of human gene promoter regions have demonstrated an overrepresentation of G₃X_<it>n</it>1G3X_<it>n</it>2G₃X_<it>n</it>3G₃motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data.</p> <p>Results</p> <p>We have used all quadruplex DNA structures deposited in the Nucleic Acid Database in order to select a list of fragments entailing a strand of three adjacent G's paired with another strand of three adjacent G's separated by a loop of one to four residues. These fragments were further clustered and representative fragments were finally selected. Further fragments were generated by assemblying the two strands of each fragment with loops from different fragments whenever the anchor G's were superimposable. The fragments were used to assemble G quadruplex based on a superimposability criterion.</p> <p>Conclusion</p> <p>Molecular models have been generated for a large number of G₃X_<it>n</it>1G₃X_<it>n</it>2G3X_<it>n</it>3G₃sequences. For a given sequence not all topologies are possible with the available repertoire of fragments due to steric hindrance and low superimposability. Since all molecular models are generated by fragments coming from observed quadruplex structures, molecular models are in principle reliable and may be used for interpretation of experimental data. Some examples of applications are given.</p

Probing the influence of citrate-capped gold nanoparticles on an amyloidogenic protein

Nanoparticles (NPs) are known to exhibit distinct physical and chemical properties compared with the same materials in bulk form. NPs have been repeatedly reported to interact with proteins, and this interaction can be exploited to affect processes undergone by proteins, such as fibrillogenesis. Fibrillation is common to many proteins, and in living organisms, it causes tissue-specific or systemic amyloid diseases. The nature of NPs and their surface chemistry is crucial in assessing their affinity for proteins and their effects on them. Here we present the first detailed structural characterization and molecular mechanics model of the interaction between a fibrillogenic protein, \u3b22-microglobulin, and a NP, 5 nm hydrophilic citrate-capped gold nanoparticles. NMR measurements and simulations at multiple levels (enhanced sampling molecular dynamics, Brownian dynamics, and Poisson-Boltzmann electrostatics) explain the origin of the observed protein perturbations mostly localized at the amino-terminal region. Experiments show that the protein-NP interaction is weak in the physiological-like, conditions and do not induce protein fibrillation. Simulations reproduce these findings and reveal instead the role of the citrate in destabilizing the lower pH protonated form of \u3b22-microglobulin. The results offer possible strategies for controlling the desired effect of NPs on the conformational changes of the proteins, which have significant roles in the fibrillation process

The significance of duodenal mucosal atrophy in patients with common variable immunodeficiency: a clinical and histopathological study

Gastrointestinal manifestations and villous atrophy can be seen in patients with common variable immunodeficiency (CVID). In some patients, infectious agents may be responsible, whereas in Others, celiac diseases (CD) may be the cause. In this study, we investigate the causes and th ehistopathologic festures seen in patients with CVID. Eleven patients with CVID and villous atrophy underwent duodenal biopsies, human leukocyte antien (HLA) typing, and testing for all celiac antibodies. Fifteen patients with CVID and normal villi and 6 patients with CD but without CVID served as controls. Histologic response to a gluten-free diet (GFD) allowed a diagnosis of CD in 3 of 11 patients. In the remaining 8, the lack of a histologic response to a GFD or HLA typing excluded CD. Celiac antibodies gave conflicting results and were of no help. Polymorphonuclear infiltrates and lesions like graft-versus-host disease are seen more ofter in flat mucos aunresponsive to a GFD. However, the specificity of these findings remains to be determined and response to a GFD remains the only diagnostic criteria for CD in these patients. Villous atrophy was gluten-sensitive in 3 of 11 patients with CVID. It was not related to gluten-responsive CD in most patients

Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications