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One of the definitions of hydrophobic interactions is the aggregation of nonpolar particles in a polar solvent, such as water. While this phenomenon appears to be very simple, it is crucial for many complex processes, such as protein folding, to take place. In this work, the hydrophobic association of adamantane and hexane at various temperatures and ionic strengths was studied using molecular dynamics simulations with the AMBER 16.0 program and the GAFF force field. The potentials of mean force of hydrophobic dimer formation, as well as the excess free energy, excess energy, excess entropy, and excess heat capacity corresponding to the formation of the contact minimum, were determined and analyzed. For both systems, the depth of the contact minimum in the potential of mean force was found to increase with both temperature and ionic strength. The excess heat capacity of the association at the contact minimum and T = 298 K was found to be negative and to decrease, while the excess entropy and energy were found to be positive and to increase for both systems, the changes being more pronounced for the hexane dimer. The excess heat capacity is also greater in absolute value for the hexane dimer.

Degradation of misfolded, redundant and oxidatively damaged proteins constitutes one of the cellular processes which are influenced by the 20S proteasome. However, its activity is generally thought to decrease with age which leads to the gradual accumulation of abnormal proteins in cells and their subsequent aggregation. Therefore, increasing proteasomal degradation constitutes a promising strategy to delay the onset of various age-related diseases, including neurodegenerative disorders. In this study we designed and obtained a series of peptidomimetic stimulators of 20S comprising in their sequences the

We present the results for CAPRI Round 50, the fourth joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of twelve targets, including six dimers, three trimers, and three higher-order oligomers. Four of these were easy targets, for which good structural templates were available either for the full assembly, or for the main interfaces (of the higher-order oligomers). Eight were difficult targets for which only distantly related templates were found for the individual subunits. Twenty-five CAPRI groups including eight automatic servers submitted ~1250 models per target. Twenty groups including six servers participated in the CAPRI scoring challenge submitted ~190 models per target. The accuracy of the predicted models was evaluated using the classical CAPRI criteria. The prediction performance was measured by a weighted scoring scheme that takes into account the number of models of acceptable quality or higher submitted by each group as part of their five top-ranking models. Compared to the previous CASP-CAPRI challenge, top performing groups submitted such models for a larger fraction (70–75%) of the targets in this Round, but fewer of these models were of high accuracy. Scorer groups achieved stronger performance with more groups submitting correct models for 70–80% of the targets or achieving high accuracy predictions. Servers performed less well in general, except for the MDOCKPP and LZERD servers, who performed on par with human groups. In addition to these results, major advances in methodology are discussed, providing an informative overview of where the prediction of protein assemblies currently stands., Cancer Research UK, Grant/Award Number: FC001003; Changzhou Science and Technology Bureau, Grant/Award Number: CE20200503; Department of Energy and Climate Change, Grant/Award Numbers: DE-AR001213, DE-SC0020400, DE-SC0021303; H2020 European Institute of Innovation and Technology, Grant/Award Numbers: 675728, 777536, 823830; Institut national de recherche en informatique et en automatique (INRIA), Grant/Award Number: Cordi-S; Lietuvos Mokslo Taryba, Grant/Award Numbers: S-MIP-17-60, S-MIP-21-35; Medical Research Council, Grant/Award Number: FC001003; Japan Society for the Promotion of Science KAKENHI, Grant/Award Number: JP19J00950; Ministerio de Ciencia e Innovación, Grant/Award Number: PID2019-110167RB-I00; Narodowe Centrum Nauki, Grant/Award Numbers: UMO-2017/25/B/ST4/01026, UMO-2017/26/M/ST4/00044, UMO-2017/27/B/ST4/00926; National Institute of General Medical Sciences, Grant/Award Numbers: R21GM127952, R35GM118078, RM1135136, T32GM132024; National Institutes of Health, Grant/Award Numbers: R01GM074255, R01GM078221, R01GM093123, R01GM109980, R01GM133840, R01GN123055, R01HL142301, R35GM124952, R35GM136409; National Natural Science Foundation of China, Grant/Award Number: 81603152; National Science Foundation, Grant/Award Numbers: AF1645512, CCF1943008, CMMI1825941, DBI1759277, DBI1759934, DBI1917263, DBI20036350, IIS1763246, MCB1925643; NWO, Grant/Award Number: TOP-PUNT 718.015.001; Wellcome Trust, Grant/Award Number: FC001003

International audience; Epidemiological studies established an association between chronic inflammation and higher risk of cancer. Inhibition of proteolytic enzymes represents a potential treatment strategy for cancer and prevention of cancer metastasis. Cathepsin C (CatC) is a highly conserved lysosomal cysteine dipeptidyl aminopeptidase required for the activation of pro-inflammatory neutrophil serine proteases (NSPs, elastase, proteinase 3, cathepsin G and NSP-4). NSPs are locally released by activated neutrophils in response to pathogens and non-infectious danger signals. Activated neutrophils also release neutrophil extracellular traps (NETs) that are decorated with several neutrophil proteins, including NSPs. NSPs are not only NETs constituents but also play a role in NET formation and release. Although immune cells harbor large amounts of CatC, additional cell sources for this protease exists. Upregulation of CatC expression was observed in different tissues during carcinogenesis and correlated with metastasis and poor patient survival. Recent mechanistic studies indicated an important interaction of tumor-associated CatC, NSPs, and NETs in cancer development and metastasis and suggested CatC as a therapeutic target in a several cancer types. Cancer cell-derived CatC promotes neutrophil recruitment in the inflammatory tumor microenvironment. Because the clinical consequences of genetic CatC deficiency in humans resulting in the elimination of NSPs are mild, small molecule inhibitors of CatC are assumed as safe drugs to reduce the NSP burden. Brensocatib, a nitrile CatC inhibitor is currently tested in a phase 3 clinical trial as a novel anti-inflammatory therapy for patients with bronchiectasis. However, recently developed CatC inhibitors possibly have protective effects beyond inflammation. In this review, we describe the pathophysiological function of CatC and discuss molecular mechanisms substantiating pharmacological CatC inhibition as a potential strategy for cancer treatment.

The recent NEWCT-9P version of the coarse-grained UNRES force field for proteins, with scale-consistent formulas for the local and correlation terms, has been tested in the CASP13 experiment of the blind-prediction of protein structure, in the ab initio, contact-assisted, and data-assisted modes. Significant improvement of the performance has been observed with respect to the CASP11 and CASP12 experiments (by over 10 GDT_TS units for the ab initio mode predictions and by over 15 GDT_TS units for the contact-assisted prediction, respectively), which is a result of introducing scale-consistent terms and improved handling of contact-distance restraints. As in previous CASP exercises, UNRES ranked higher in the free modeling category than in the general category that included template based modeling targets. Use of distance restraints from the predicted contacts, albeit many of them were wrong, resulted in the increase of GDT_TS by over 8 units on average and introducing sparse restraints from small-angle X-ray/neutron scattering and chemical cross-link-mass-spectrometry experiments, and ambiguous restraints from nuclear magnetic resonance experiments has also improved the predictions by 8.6, 9.7, and 10.7 GDT_TS units on average, respectively.

Rat sialorphin (Gln-His-Asn-Pro-Arg) is a natural blocker of neprilysin (NEP) that belongs to the family of endogenous opioid peptide-degrading enzymes. Studies have confirmed the efficiency of sialorphin in blocking the activity of NEP, both in vitro and in vivo. It has been demonstrated that this inhibitor has a strong analgesic, anti-inflammatory, immunological and metabolic effect either directly or indirectly by affecting the level of Met/Leu-enkephalins. In this work, sialorphin and their 12 analogues were synthesised using the solid-phase method. The effect of the peptides on the degradation of Met-enkephalin by NEP and metabolic degradation in human plasma was investigated in vitro. We show that the change in the N-terminal amino acid configuration from L to D in almost all peptides, except D-Arg-His-Asn-Pro-Arg (peptide XI), led to the abolition of their inhibitory activity. With molecular modelling technique we explained the structural properties of the L and D-arginine located on the N-terminal part of the peptide. The detailed analysis of the protein binding pocket allowed us to explain why D-arginine is so unique among all D residues. Peptide XI showed the highest stability among the tested peptides in human plasma. For instance sialorphin after a 2-hour incubation in human plasma was almost completely decomposed, while the level of peptide XI dropped to 45% after 48 h under these conditions.

International audience; Cathepsin C (CatC) is a dipeptidyl-exopeptidase which activates neutrophil serine protease precursors (elastase, proteinase 3, cathepsin G and NSP4) by removing their N-terminal propeptide in bone marrow cells at the promyelocytic stage of neutrophil differentiation. The resulting active proteases are implicated in chronic inflammatory and autoimmune diseases. Hence, inhibition of CatC represents a therapeutic strategy to suppress excessive protease activities in various neutrophil mediated diseases. We designed and synthesized a series of dipeptidyl cyclopropyl nitrile compounds as putative CatC inhibitors. One compound, IcatCXPZ-01 ((S)-2-amino-N-((1R,2R)-1-cyano-2-(4'-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-yl)cyclopropyl)butanamide)) was identified as a potent inhibitor of both human and rodent CatC. In mice, pharmacokinetic studies revealed that IcatCXPZ-01 accumulated in the bone marrow reaching levels suitable for CatC inhibition. Subcutaneous administration of IcatCXPZ-01 in a monoclonal anti-collagen antibody induced mouse model of rheumatoid arthritis resulted in statistically significant anti-arthritic activity with persistent decrease in arthritis scores and paw thickness.

The UNited RESidue (UNRES) force field was tested in the 14th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP14), in which larger oligomeric and multimeric targets were present compared to previous editions. Three prediction modes were tested (i) ab initio (the UNRES group), (ii) contact-assisted (the UNRES-contact group), and (iii) template-assisted (the UNRES-template group). For most of the targets, the contact restraints were derived from the server models top-ranked by the DeepQA method, while the DNCON2 method was used for 11 targets. Our consensus-fragment procedure was used to run template-assisted predictions. Each group also processed the Nuclear Magnetic Resonance (NMR)- and Small Angle X-Ray Scattering (SAXS)-data assisted targets. The average Global Distance Test Total Score (GDT_TS) of the ‘Model 1’ predictions were 29.17, 39.32, and 56.37 for the UNRES, UNRES-contact, and UNRES-template predictions, respectively, increasing by 0.53, 2.24, and 3.76, respectively, compared to CASP13. It was also found that the GDT_TS of the UNRES models obtained in ab initio mode and in the contact-assisted mode decreases with the square root of chain length, while the exponent in this relationship is 0.20 for the UNRES-template group models and 0.11 for the best performing AlphaFold2 models, which suggests that incorporation of database information, which stems from protein evolution, brings in long-range correlations, thus enabling the correction of force-field inaccuracies.

This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (&beta, 2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ&ndash, Dap(O2(Cbz))&ndash, Dap(GO1)&ndash, Arg&ndash, ANB&ndash, NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 &plusmn, 0.02 &mu, M, kcat = 245 s&minus, 1, and kcat/Km = 7.61 &times, 107 M&minus, 1 s&minus, 1. This process was practically halted when a selective inhibitor of the &beta, 2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10&minus, 11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.

The method for protein-structure prediction, which combines the physics-based coarse-grained UNRES force field with knowledge-based modeling, has been developed further and tested in the 13th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP13). The method implements restraints from the consensus fragments common to server models. In this work, the server models to derive fragments have been chosen on the basis of quality assessment; a fully automatic fragment-selection procedure has been introduced, and Dynamic Fragment Assembly pseudopotentials have been fully implemented. The Global Distance Test Score (GDT_TS), averaged over our "Model 1" predictions, increased by over 10 units with respect to CASP12 for the free-modeling category to reach 40.82. Our "Model 1" predictions ranked 20 and 14 for all and free-modeling targets, respectively (upper 20.2% and 14.3% of all models submitted to CASP13 in these categories, respectively), compared to 27 (upper 21.1%) and 24 (upper 18.9%) in CASP12, respectively. For oligomeric targets, the Interface Patch Similarity (IPS) and Interface Contact Similarity (ICS) averaged over our best oligomer models increased from 0.28 to 0.36 and from 12.4 to 17.8, respectively, from CASP12 to CASP13, and top-ranking models of 2 targets (H0968 and T0997o) were obtained (none in CASP12). The improvement of our method in CASP13 over CASP12 was ascribed to the combined effect of the overall enhancement of server-model quality, our success in selecting server models and fragments to derive restraints, and improvements of the restraint and potential-energy functions.

Based on the coarse-grained UNRES and NARES-2P models of proteins and nucleic acids, respectively, developed in our laboratory, in this work we have developed a coarse-grained model of systems containing proteins and nucleic acids. The UNRES and NARES-2P effective energy functions have been applied to the protein and nucleic-acid components of a system, respectively, while protein-nucleic-acid interactions have been described by the respective coarse-grained potentials developed in our recent work (Yin et al., J. Chem Theory Comput. 2015, 11, 1792). The Debye-Huckel screening has been applied to the electrostatic-interaction energy between the phosphate groups and charged amino-acid side chains. The model has been integrated into the UNRES package for coarse-grained molecular dynamics simulations of proteins and the implementation has been tested for energy conservation in microcanonical molecular dynamics runs and for temperature conservation in canonical molecular dynamics runs. Two case studies were performed: (i) the dynamics of the Ku protein heterodimer bound to DNA, for which it was found that the Ku70/Ku80 protein complex plays an active role in DNA repairing and (ii) conformational changes of the multiple antibiotic resistance (MarA) protein occurring during DNA binding, for which the functionally important motions occurring during this process were identified. © 2018 Wiley Periodicals, Inc.

Enkephalins are involved in a number of physiological processes. However, these peptides are quickly degraded by peptidases, e.g. the neutral endopeptidase (NEP). Inhibition of the enzymatic degradation of enkephalins is one of the possible approaches to prolong their activity. Selective inhibitor of NEP, sialorphin, is the attractive lead compound for enkephalins degradation studies. In this work, an alanine scan of sialorphin and a series of its hybrids with opiorphin, synthesised by the solid phase method, were performed. The effect of the peptides on degradation of Met-enkephalin by NEP in vitro was investigated. Molecular modelling technique was used to identify residues responsible for protein–ligand interactions. We showed that substitution of amino acids Gln1, Pro4 and Arg5 of sialorphin for Ala significantly reduced the half-life of Met-enkephalin in the presence of NEP. [Ala3]sialorphin displayed a higher inhibitory potency against NEP than sialorphin. Substitution of His2 for Ala led to a compound which was as active as lead compound. Sialorphin has a structure which hardly tolerates substitution in its sequence at positions 1, 4 and 5. The conversion of His2 for alanine in sialorphin is tolerated very well. The higher inhibitory potency of [Ala3]sialorphin than sialorphin against NEP is caused by removal of the hydrophilic residue (Asn) and a better fit of the peptide to the enzyme-binding pocket. The role of side chains of sialorphin in degradation of enkephalin by NEP has been explored. This study also provides an important SAR information essential for further drug design.

One of the possible applications of ionic liquids is to produce electricity from heat. The iron oxide nanoparticle is a potent electrical particle, which is expected to improve the heat's efficiency to electricity conversion, however, it is prone to aggregation and sedimentation, which hamper its application. One of the methods to enhance the nanoparticle's solubility and electrical properties is the use of a stabilizing component in the production and application process. In this research, we studied, how the nanoparticle affects the properties of the 1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMIM TFSI) ionic liquid. The citric acid was used as a nanoparticle stabilizing agent and led to decrease of the diffusion coefficient of the ionic liquid's components. Our results indicated, that with the addition of the citric acid, strong solvation sphere around nanoparticle was formed. The presence of nanoparticles significantly reduced the diffusion of ionic liquid components. Finally, the citric acid was found to be the strongest nanoparticle binding component of the studied system, and therefore it was able to stabilize the nanoparticle in solution.

Experimental methods of investigations of nanoparticle (NP)–protein interactions are limited, because they require a high amount of samples and the NPs tend to interfere with spectral results. Therefore, molecular modeling is a commonly accepted tool in such kind of investigations. Examining the molecule toxicity on the molecular level, we usually want to know, mainly, the location of the ligand on the protein surface and what is an influence of such a contact on the biological functions of the protein. In the presented work, we demonstrate that multiple-docking of the ligand from a random start and with large grid volume, to let the ligand search the whole protein surface, allows to find the best binding sites and gives reliable results considering ligand–protein interactions. In the present work, we have constructed six models of bronchoalveolar lavage fluids proteins: α1-antitripsin, albumin, ceruloplasmin, lactoferrin, lysozyme, and transferrin with fullerene, C60 utilizing molecular docking methods. The most probable results were examined with steered molecular dynamics (SMD) to see, if the simple docking method is able to predict the fullerene binding affinity. Albumin and lysozyme were already widely investigated and literature data is available for their complexes with fullerene C60 and/or its derivatives. Thus, we used these two models as a reference set to validate the used molecular modeling methods. With our best knowledge, interactions of the remaining four proteins with NPs have never been investigated in detail before. Our results indicate that fullerene C60 readily interacts with all studied proteins and may have a large impact on their biological functions.

Ionic liquids (ILs) have attracted much attention for their unique physicochemical properties, which can be designed as needed by altering the ion combinations. Besides experimental work, numerous computational studies have been concerned with prediction of physical properties of ILs. The results of molecular dynamics simulations of ILs depend strongly on the proper force field parameterization. Classical force fields and the force fields designed specifically for ILs have been found to yield diffusion coefficients, that are too low for the liquid state. This has been attributed to the lack of electronic polarizability in the most force fields. One simple solution to this problem has been uniform by scaling down of the partial charges of all ions. In this work we investigated the influence of the charge scaling, the size of the simulated system and the temperature factor on calculated density, radial distribution function and the diffusion coefficients of the cation and anion of two methylpyridinium based ionic liquids: 1-butyl-4-methylpyridinium tetrafluoroborate ([b4mpy][BF 4 ]) and 1-butyl-4-methylpyridinium chloride ([b4mpy][Cl]). We show that the parameterization is the key for a proper reproduction of the diffusion coefficient and as a consequence the melting temperature and ionic conductivity. We were also able to get some structural informations on the cation-anion relationships in the investigated ILs.

The proteasome degrade most of the proteins in eukaryotic cells, thereby controlling the keycellular processes [...]

Phosphorylated proteins take part in many signaling pathways and play a key role in homeostasis regulation. The all-atom force fields enable us to study the systems containing phosphorylated proteins, but they are limited to short time scales. In this paper, we report the extension of the physics-based coarse-grained UNRES force field to treat systems with phosphorylated amino-acid residues. To derive the respective potentials, appropriate physics-based analytical expressions were fitted to the potentials of mean force of systems modeling phosphorylated amino-acid residues computed in our previous work and implemented in UNRES. The extended UNRES performed well in ab initio simulations of two miniproteins containing phosphorylated residues, strongly suggesting that realistic large-scale simulations of processes involving phosphorylated proteins, especially signaling processes, are now possible.

In this paper we present the methodology for assessing the ionic liquids’ viscosity at six temperature points (25, 35, 45, 50, 60 and 70 [°C]), which utilizes only the in silico approach. The main idea of such assessment is based on the “correction equation” describing the correlation between experimentally measured viscosity and theoretically derived density (calculated with use of molecular mechanics), given at 6 different temperature points. The equation allows for recalculating the viscosity of ILs at 25 °C to determine the viscosity of ILs at other, higher temperature. Since the equation needs the basic viscosity value (at 25 °C) we additionally developed QSPR model that allows predicting it. According to our model, the viscosity of ILs is dependent to the size and branching of the cation and size, shape, symmetry and the vertical electron binding energy of IL’s anion. With those novel tools, it is possible to predict the viscosity of new ionic liquids at different temperatures without the need of experimental measurements.

Summary: Participating as the Cornell-Gdansk group, we have used our physics-based coarse-grained UNited RESidue (UNRES) force field to predict protein structure in the 11th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP11). Our methodology involved extensive multiplexed replica exchange simulations of the target proteins with a recently improved UNRES force field to provide better reproductions of the local structures of polypeptide chains. All simulations were started from fully extended polypeptide chains, and no external information was included in the simulation process except for weak restraints on secondary structure to enable us to finish each prediction within the allowed 3-week time window. Because of simplified UNRES representation of polypeptide chains, use of enhanced sampling methods, code optimization and parallelization and sufficient computational resources, we were able to treat, for the first time, all 55 human prediction targets with sizes from 44 to 595 amino acid residues, the average size being 251 residues. Complete structures of six single-domain proteins were predicted accurately, with the highest accuracy being attained for the T0769, for which the CαRMSD was 3.8 Å for 97 residues of the experimental structure. Correct structures were also predicted for 13 domains of multi-domain proteins with accuracy comparable to that of the best template-based modeling methods. With further improvements of the UNRES force field that are now underway, our physics-based coarse-grained approach to protein-structure prediction will eventually reach global prediction capacity and, consequently, reliability in simulating protein structure and dynamics that are important in biochemical processes. Availability and Implementation: Freely available on the web at http://www.unres.pl/ . Contact: has5@cornell.edu

HtrA2(Omi) protease is involved in the maintenance of mitochondrial homeostasis and stimulation of apoptosis as well as in development of cancer and neurodegenerative disorders. The protein is a homotrimer whose subunits comprise serine protease domain (PD) and PDZ regulatory domain. In the basal, inactive state, a tight interdomain interface limits access both to the PDZ peptide (carboxylate) binding site and to the PD catalytic center. The molecular mechanism of activation is not well understood. To further the knowledge of HtrA2 thermal activation we monitored the dynamics of the PDZ-PD interactions during temperature increase using tryptophan-induced quenching (TrIQ) method. The TrIQ results suggested that during activation the PDZ domain changed its position versus PD inside a subunit, including a prominent change affecting the L3 regulatory loop of PD, and also changed its interactions with the PD of the adjacent subunit (PD*), specifically with its L1* regulatory loop containing the active site serine. The α5 helix of PDZ was involved in both, the intra- and intersubunit changes of interactions and thus seems to play an important role in HtrA2 activation. The amino acid substitutions designed to decrease the PDZ interactions with the PD or PD* promoted protease activity at a wide range of temperatures, which supports the conclusions based on the TrIQ analysis. The model presented in this work describes PDZ movement in relation to PD and PD*, resulting in an increased access to the peptide binding and active sites, and conformational changes of the L3 and L1* loops.

Every two years groups worldwide participate in the Critical Assessment of Protein Structure Prediction (CASP) experiment to blindly test the strengths and weaknesses of their computational methods. CASP has significantly advanced the field but many hurdles still remain, which may require new ideas and collaborations. In 2012 a web-based effort called WeFold, was initiated to promote collaboration within the CASP community and attract researchers from other fields to contribute new ideas to CASP. Members of the WeFold coopetition (cooperation and competition) participated in CASP as individual teams, but also shared components of their methods to create hybrid pipelines and actively contributed to this effort. We assert that the scale and diversity of integrative prediction pipelines could not have been achieved by any individual lab or even by any collaboration among a few partners. The models contributed by the participating groups and generated by the pipelines are publicly available at the WeFold website providing a wealth of data that remains to be tapped. Here, we analyze the results of the 2014 and 2016 pipelines showing improvements according to the CASP assessment as well as areas that require further adjustments and research. Funding Agencies|Office of Science of the U.S. Department of Energy [DE-AC02-05CH11231]; U.S. Department of Energy, Office of Science, Office of Workforce Development for Teachers and Scientists (WDTS) under the Science Undergraduate Laboratory Internship (SULI) program; U.S. Department of Energy, Office of Science, Office of Workforce Development for Teachers and Scientists (WDTS) under the Visiting Faculty Program (VFP); United States-Israel Binational Science Foundation (BSF) [2009432]; Israel Science Foundation (ISF) [1122/14]; National Institute of General Medical Sciences [R01GM093123, GM083107, GM116960]; Purdue University start-up funds; Ralph W. and Grace M. Showalter Trust Award; Jim and Diann Robbers Cancer Research Grant for New Investigators Award; Brazilian agency: FAPESP; Brazilian agency: CAPES; Brazilian agency: CNPq; NIH [GM-14312]; NSF [MCB-10-19767]; National Institutes of Medicine [GM11574901]; Swedish Research Council [2012-5270, 2016-05369]; Swedish e-Science Research Center; Polish National Science Center [UMO-2013/10/M/ST4/00640]; IISc Mathematical Initiative Assistantship; National Academy of Sciences, India; National Institutes of Health [R01-GM100701, R01GM052032]; National Science Foundation; National Science Foundation Graduate Research Fellowship [DGE-1148900]; Princeton Institute for Computational Science and Engineering (PICSciE); Princeton University Office of Information Technology; UK Engineering and Physical Sciences Research Council [EP/M020576/1, EP/N031962/1]; National Research Foundation of Korea [2016R1A2A1A05005485]

Based on the coarse-grained UNRES and NARES-2P models of proteins and nucleic acids, respectively, developed in our laboratory, in this work we have developed a coarse-grained model of systems containing proteins and nucleic acids. The UNRES and NARES-2P effective energy functions have been applied to the protein and nucleic-acid components of a system, respectively, while protein-nucleic-acid interactions have been described by the respective coarse-grained potentials developed in our recent work (Yin et al., J. Chem Theory Comput. 2015, 11, 1792). The Debye-Hückel screening has been applied to the electrostatic-interaction energy between the phosphate groups and charged amino-acid side chains. The model has been integrated into the UNRES package for coarse-grained molecular dynamics simulations of proteins and the implementation has been tested for energy conservation in microcanonical molecular dynamics runs and for temperature conservation in canonical molecular dynamics runs. Two case studies were performed: (i) the dynamics of the Ku protein heterodimer bound to DNA, for which it was found that the Ku70/Ku80 protein complex plays an active role in DNA repairing and (ii) conformational changes of the multiple antibiotic resistance (MarA) protein occurring during DNA binding, for which the functionally important motions occurring during this process were identified. © 2018 Wiley Periodicals, Inc.

Helicobacter pylori HP0377 is a thiol oxidoreductase, a member of the CcmG family involved in cytochrome biogenesis, as previously shown by in vitro experiments. In this report, we document that HP0377 also acts in vivo in the cytochrome assembly process in Bacillus subtilis, where it complements the lack of ResA. However, unlike other characterized proteins in this family, HP0377 is a dithiol reductase and isomerase. We elucidated how the amino acid composition of its active site modulates its functionality. We demonstrated that cis-proline (P156) is involved in its interaction with the redox partner (CcdA), as a P156T HP0377 variant is inactive in vivo and is present in the oxidized form in B. subtilis. Furthermore, we showed that engineering the HP0377 active motif by changing CSYC motif into CSYS or SSYC, clearly diminishes two activities (reduction and isomerization) of the protein. Whereas HP0377CSYA is inactive in reduction as well as in isomerization, HP0377CSYS retains reductive activity. Also, replacement of F95 by Q decreases its ability to regenerate scRNase and does not influence the reductive activity of HP0377CSYS towards apocytochrome c. HP0377 is also distinguished from other CcmGs as it forms a 2:1 complex with apocytochrome c. Phylogenetic analyses showed that, although HP0377 is capable of complementing ResA in Bacillus subtilis, its thioredoxin domain has a different origin, presumably common to DsbC.

Knowledge-based methods are, at present, the most effective ones for the prediction of protein structures; however, their results heavily depend on the similarity of a target sequence to those of proteins with known structures. On the other hand, the physics-based methods, although still less accurate and more expensive to execute, are independent of databases and give reasonable results where the knowledge-based methods fail because of weak sequence similarity. Therefore, a plausible approach seems to be the use of knowledge-based methods to determine the sections of the structures that correspond to sufficient sequence similarity and physics-based methods to determine the remaining structure. By participating in the 12th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP12) as the KIAS-Gdansk group, we tested our recently developed hybrid approach, in which protein-structure prediction is carried out by using the physics-based UNRES coarse-grained energy function, with restraints derived from the server models. Best predictions among all groups were obtained for 2 targets and 80% of our models were in the upper 50% of the models submitted to CASP. Our method was also able to exclude, with about 70% confidence, the information from the servers that performed poorly on a given target. Moreover, the method resulted in the best models of 2 refinement targets and performed remarkably well on oligomeric targets.

The p-xylylene monomers of parylene N, C and D have similar high polymerization reactivity. For effective copolymerization processes this fact is basically a drawback and for instance the copolymerization with styrene doesn’t go at all (Corley et al. J Pol Sc 13(68):137–156, [15]). Substitution of terminal hydrogen atoms by chlorine atoms reduces reactivity dramatically. 7,7,8,8-tetrachloro-p-xylylene and 2,5,7,7,8,8-hexachloro-p-xylylene can be isolated as yellow crystals. These crystals can be kept without any change in temperature below 0 ∘C, but they polymerize slowly at room temperature. Perchloro-p-xylylene is stable even at elevated temperatures and does not polymerize under any conditions. Both 7,7,8,8-tetrachloro-p-xylylene and 2,5,7,7,8,8-hexachloro-p-xylylene copolymerize with various vinyl monomers, such as styrene and others. In this work the polymerization reactions of different chloro-derivatives of p-xylylene were modeled by means of the DFT method with hybrid correlation functionals (B3LYP and PBE0) and, for comparison, by means of the Hartree Fock methods. We inquired both initiation as well as elongation polymeric reactions for each of the reactants. We survied their reactivity analytically examining energetics and configurations in Szwarc-like process. The quantitative influence of chlorine atoms on the reactivity in polymerization steps, their location in the reactants’ structure (aromatic and/or aliphatic) as well as their number, were reviewed. The polymerizations of p-xylylenes with chlorine atoms as terminal aliphatic substituents yet revealed one more access path for parylenes’ in situ functionalization. Electronic supplementary material The online version of this article (doi:10.1007/s00894-016-3179-6) contains supplementary material, which is available to authorized users.

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA′ (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.

Aim: The pharmacotherapy of inflammatory bowel disease is difficult and currently available treatments bring mostly poor and unsatisfactory results. Results: The purpose of this work was the synthesis of opiorphin, sialorphin, spinorphin and a series of their analogs and the in vitro characterization of their effect on degradation of enkephalin by neutral endopeptidase and aminopeptidase N. Consequently, we investigated in vivo the anti-inflammatory effect of the most active inhibitors selected in the in vitro studies (Pal-KKQRFSR & Pal-KKQHNPR). Putative inhibitor – enzyme (neutral endopeptidase or aminopeptidase N) complexes are also presented and their binding interfaces are identified. Conclusion: Our results suggest that Pal-KKQHNPR has the potential to become a valuable template for anti-inflammatory therapeutics for the treatment of GI tract inflammation.

The folding of the N-terminal part of the B-domain of staphylococcal protein A (PDB ID: 1BDD, a 46-residue three-α-helix bundle) and the formin-binding protein 28 WW domain (PDB ID: 1E0L, a 37-residue three-stranded anti-parallel β protein) was studied by means of Langevin dynamics with the coarse-grained UNRES force field to assess the influence of hydrodynamic interactions on protein-folding pathways and kinetics. The unfolded, intermediate, and native-like structures were identified by cluster analysis, and multi-exponential functions were fitted to the time dependence of the fractions of native and intermediate structures, respectively, to determine bulk kinetics. It was found that introducing hydrodynamic interactions slows down both the formation of an intermediate state and the transition from the collapsed structures to the final native-like structures by creating multiple kinetic traps. Therefore, introducing hydrodynamic interactions considerably slows the folding, as opposed to the results obtained from earlier studies with the use of Gō-like models.

High-temperature requirement A (HtrA; DegP) from Escherichia coli, an important element of the extracytoplasmic protein quality-control system, is a member of the evolutionarily conserved family of serine proteases. The characteristic feature of this protein is its allosteric mode of activation. The regulatory loops, L3, L2, L1 and LD, play a crucial role in the transmission of the allosteric signal. Yet, the role of LD has not been fully elucidated. Therefore, we undertook a study to explain the role of the individual LD residues in inducing and maintaining the proteolytic activity of HtrA. We investigated the influence of amino acid substitutions located within the LD loop on the kinetics of a model substrate cleavage as well as on the dynamics of the oligomeric structure of HtrA. We found that the mutations that were expected to disturb the loop's structure and/or interactions with the remaining regulatory loops severely diminished the proteolytic activity of HtrA. The opposite effect, that is, increased activity, was observed for G174S substitution, which was predicted to strengthen the interactions mediated by LD. HtrAG174S protein had an equilibrium shifted toward the active enzyme and formed preferentially high-order oligomeric forms.

Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.

HtrA2(Omi) protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2S306A mutant (with the catalytic serine routinely disabled for an X-ray study to avoid self-degradation) is a homotrimer whose subunits contain the serine protease domain (PD) and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD). V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2), were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D, but does not allow to explain an increased activity of HtrA2V226K.

The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperature range 25-45 degrees C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes (tau) and wavelength emission maxima (lambda(em)max) were correlated with the induction of the HtrA proteolytic activity. Generally the temperature shift caused better exposure of Trps to the quencher; although, each of the loops was affected differently. The LA loop seemed to be the most prone to temperature-induced conformational changes and a significant opening of its structure was observed even at the lowest temperatures tested (25-30 degrees C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 degrees C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 degrees C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity.

S100B protein is one of the factors involved in the down-regulation of tumor suppressor protein p53, a transcription activator that signals for cycle arrest and apoptosis. As the inactivation of normal p53 functions is found in over half of human cancers, restoration of normal p53 functions through the destruction or prevention of S100B - p53 complexes represents a possible approach for the development of anti-cancer drugs. The aim of this work was to propose the S100B binding interface through an examination of the literature and use of molecular modeling (MM) techniques with AutoDock program and the AMBER force field. We propose two residues in the S100B binding pocket (Val56, Phe76) and two residues on the protein surface (Val52, Ala83) are essential for ligand binding. The data presented here indicate that interactions with these four residues are necessary for a reduction in the incidence of the S100B - p53 complex. Additionally, we have tried to explain a mechanism for the action of pentamidine, the best-known S100B ligand, and have proposed two S100B - pentamidine structures. The results presented here may be useful for the efficient design of new S100B ligands.

For many years RasMol was one of the most used programs for molecular visualization. It was an excellent tool due to its simplicity and its low demand of computer power. Today it is replaced by OpenGL programs, which have excellent graphics that new computers can additionally handle. Molecular graphics is one of the best tools for the analysis of biomolecular data. With high efficiency and a low demand of computer power, RasMol can still be used as a quick and handy tool used for the analysis of biomolecular structures with good results. In this paper, we describe modifications to the RasMol program, as implemented on the base of RasMol AB 2. We introduced several new functions, namely: the identification of histidine isomers, and advanced structural selection and macro capabilities (as implemented in the point-click menu), which result in an increase in the speed and accuracy of structural analyses. The program can be downloaded from the project page: http://etoh.chem.univ.gda.pl/rasmol/.

The potentials of mean force (PMFs) were determined for systems forming molecular and ionic heterocomplexes (in which the proton is transferred from a neutral acid to a neutral base molecule) composed of acetic acid and phenol with amines in three solvents with different polarities and hydrogen-bonding propensities: acetonitrile (AN), dimethyl sulfoxide (DMSO), and water. For each pair and each solvent, a series of umbrella-sampling molecular dynamics simulations with the AMBER force field and explicit solvent molecules were carried out and the PMF was calculated by using the weighted histogram analysis method (WHAM). Subsequently, heteroconjugation equilibrium constants were calculated by numerical integration of the respective PMF profiles. All complexes except those involving acetic acid as a proton donor in water were found to form in solution, which was manifested by the presence of contact minima corresponding to hydrogen-bonded species in the PMF curves. The calculated heteroconjugation constants were found to be greater for complexes with proton transfer than for those without proton transfer for the acetic acid-n-butylamine system in all solvents and for the phenol-imidazole and phenol-n-butylamine systems in DMSO when the same reference state (molecular acid and molecular base) was considered. For the acetic acid-n-butylamine system in acetonitrile and dimethyl sulfoxide, the calculated constants of ionic heterocomplex formation are greater than the values determined by potentiometric titration, while the calculated constants of molecular heterocomplex formation, as well as the calculated constants of heterocomplex formation from acetic acid and 4(5)-methylimidazole, are in good agreement with the experimental data, which suggests that the force field applied overestimates the energy of the interactions of oppositely charged ions or the energy of the interactions of hydrogen-bonding species with solvent molecules. The constants were found to decrease with an increasing polarity and hydrogen-bonding propensity of the solvent (i.e., in the series AN > DMSO > H 2 O), this being in agreement with the available experimental data.

Molecular docking simulations are now fast developing area of research. In this work we describe an effective procedure of preparation of the receptor-ligand complexes. The amino-acid residues involved in ligand binding were identified and described.

The nonapeptide hormones arginine vasopressin (CYFQNCPRG-NH2, AVP) and oxytocin (CYIQNCPLG-NH2, OT), control many essential functions in mammals. Their main activities include the urine concentration (via stimulation of AVP V2 receptors, V2R, in the kidneys), blood pressure regulation (via stimulation of vascular V1a AVP receptors, V1aR), ACTH control (via stimulation of V1b receptors, V1bR, in the pituitary) and labor and lactation control (via stimulation of OT receptors, OTR, in the uterus and nipples, respectively). All four receptor subtypes belong to the GTP-binding (G) protein-coupled receptor (GPCR) family. This work consists of docking of YM087, a potent non-peptide V1aR and V2R - but not OTR - antagonist, into the receptor models based on relatively new theoretical templates of rhodopsin (RD) and opiate receptors, proposed by Mosberg et al. (Univ. of Michigan, Ann Arbor, USA). It is simultaneously demonstrated that this RD template satisfactorily compares with the first historical GPCR structure of bovine rhodopsin (Palczewski et al., 2000) and that homology-modeling of V2R, V1aR and OTR using opiate receptors as templates is rational, based on relatively high (20-60%) sequence homology among the set of 4 neurophyseal and 4 opiate receptors. YM087 was computer-docked to V1aR, V2R and OTR using the AutoDock (Olson et al., Scripps Research Institute, La Jolla, USA) and subsequently relaxed using restrained simulated annealing and molecular dynamics, as implemented in AMBER program (Kollman et al., University of California, San Francisco, USA). From about 80 diverse configurations, sampled for each of the three ligand/receptor systems, 3 best energy-relaxed complexes were selected for mutual comparisons. Similar docking modes were found for the YM087/V1aR and YM087/V2R complexes, diverse from those of the YM087/OTR complexes, in agreement with the molecular affinity data.

HtrA2(Omi), belonging to the high-temperature requirement A (HtrA) family of stress proteins, is involved in the maintenance of mitochondrial homeostasis and in the stimulation of apoptosis, as well as in cancer and neurodegenerative disorders. The protein comprises a serine protease domain and a postsynaptic density of 95 kDa, disk large, and zonula occludens 1 (PDZ) regulatory domain and functions both as a protease and a chaperone. Based on the crystal structure of the HtrA2 inactive trimer, it has been proposed that PDZ domains restrict substrate access to the protease domain and that during protease activation there is a significant conformational change at the PDZ–protease interface, which removes the inhibitory effect of PDZ from the active site. The crystal structure of the HtrA2 active form is not available yet. HtrA2 activity markedly increases with temperature. To understand the molecular basis of this increase in activity, we monitored the temperature-induced structural changes using a set of single-Trp HtrA2 mutants with Trps located at the PDZ–protease interface. The accessibility of each Trp to aqueous medium was assessed by fluorescence quenching, and these results, in combination with mean fluorescence lifetimes and wavelength emission maxima, indicate that upon an increase in temperature the HtrA2 structure relaxes, the PDZ–protease interface becomes more exposed to the solvent, and significant conformational changes involving both domains occur at and above 30 °C. This conclusion correlates well with temperature-dependent changes of HtrA2 proteolytic activity and the effect of amino acid substitutions (V226K and R432L) located at the domain interface, on HtrA2 activity. Our results experimentally support the model of HtrA2 activation and provide an insight into the mechanism of temperature-induced changes in HtrA2 structure. Electronic supplementary material The online version of this article (doi:10.1007/s12192-012-0355-1) contains supplementary material, which is available to authorized users.

The mechanism of polymerization of p-xylylene and its derivatives is analyzed at the theoretical level. The polymerization reaction takes place in vacuo without any catalyst. The first step is a pyrolytic decomposition of starting material for polymerization, p-cyclophane, a cyclic dimer of p-xylylene, into biradical linear dimer and finally into two quinonoid monomeric molecules of p-xylylene. The quinonoid monomer is diamagnetic; i.e., it has a singlet ground state. The monomers after pyrolysis, when the temperature is lowered, do not re-form cyclic dimers but instead polymerize into long chain molecules. The initiation of polymerization requires dimerization of two monomers leading to formation of a genuine noncoupled biradical dimer. The chain molecules grow through the propagation reaction only one unit at a time, by the attachment of a monomer to a radical chain end. In this work the pyrolysis reaction, the initiation reaction and the first propagation steps of parylene polymerization (up to pentamer) are studied in details using different quantum chemical methods: AM1 and PM6 semiempirical methods and density functional theory (DFT) approach using B3LYP functional with two basis sets of different size (SVP and TZVP).