Set Of Restraints Research Articles

Alchemical Calculation of Relative Free Energies for Charge-Changing Mutations at Protein-Protein Interfaces Considering Fixed and Variable Protonation States.

The calculation of relative free energies (ΔΔG) for charge-changing mutations at protein-protein interfaces through alchemical methods remains challenging due to variations in the system's net charge during charging steps, the possibility of mutated and contacting ionizable residues occurring in various protonation states, and undersampling issues. In this study, we present a set of strategies, collectively termed TIRST/TIRST-H+, to address some of these challenges. Our approaches combine thermodynamic integration (TI) with the prediction of pKa shifts to calculate ΔΔG values. Moreover, special sets of restraints are employed to keep the alchemically transformed molecules separated. The accuracy of the devised approaches was assessed on a large and diverse data set comprising 164 point mutations of charged residues (Asp, Glu, Lys, and Arg) to Ala at the protein-protein interfaces of complexes with known three-dimensional structures. Mean absolute and root-mean-square errors ranging from 1.38 to 1.66 and 1.89 to 2.44 kcal/mol, respectively, and Pearson correlation coefficients of ∼0.6 were obtained when testing the approaches on the selected data set using the GPU-TI module of Amber18 suite and the ff14SB force field. Furthermore, the inclusion of variable protonation states for the mutated acid residues improved the accuracy of the predicted ΔΔG values. Therefore, our results validate the use of TIRST/TIRST-H+ in prospective studies aimed at evaluating the impact of charge-changing mutations to Ala on the stability of protein-protein complexes.

Has financial fair play changed European football?

In 2011 UEFA, the governing body of European football, introduced the Financial Fair Play Regulation (FFPR), consisting of a set of financial restraints to be met by clubs as a prerequisite for participation to its competitions. The aim of the FFPR was to introduce financial discipline into the clubs’ decision-making processes, and ultimately protect the long-term viability of the European football industry. The reform was criticized because of possible unintended detrimental consequences. In particular, Peeters and Szymanski (2014) provided a model-based ex-ante simulation analysis showing that the reform would increase the profitability of clubs, but also tilt the competitive balance in favor of the top teams, thus reducing the interest of fans and investors as one of the main attractions in sports is precisely that the best team does not always win. Exploiting an original dataset between the seasons 2007–2008 and 2019–2020, we provide an ex-post econometric evaluation of the effects of the introduction of the FFPR revealing causal evidence that largely vindicates those ex-ante predictions.

Improving the quality of co-evolution intermolecular contact prediction with DisVis.

The steep rise in protein sequences and structures has paved the way for bioinformatics approaches to predict residue-residue interactions in protein complexes. Multiple sequence alignments are commonly used in contact predictions to identify co-evolving residues. These contacts, however, often include false positives (FPs), which may impair their use to predict three dimensional structures of biomolecular complexes and affect the accuracy of the generated models. Previously, we have developed DisVis to identify FP in mass spectrometry cross-linking data. DisVis allows to assess the accessible interaction space between two proteins consistent with a set of distance restraints. Here, we investigate if a similar approach could be applied to co-evolution predicted contacts in order to improve their precision prior to using them for modeling. We analyze co-evolution contact predictions with DisVis for a set of 26 protein-protein complexes. The DisVis-reranked and the original co-evolution contacts are then used to model the complexes with our integrative docking software HADDOCK using different filtering scenarios. Our results show that HADDOCK is robust with respect to the precision of the predicted contacts due to the 50% random contact removal during docking and can enhance the quality of docking predictions when combined with DisVis filtering for low precision contact data. DisVis can thus have a beneficial effect on low quality data, but overall HADDOCK can accommodate FP restraints without negatively impacting the quality of the resulting models. Other more precision-sensitive docking protocols might, however, benefit from the increased precision of the predicted contacts after DisVis filtering.

Improving the Accuracy of Small‐Molecule Crystal Structures Solved from Powder X‐Ray Diffraction Data by Using External Sources

AbstractStructural analysis using powder X‐ray diffraction data has overcome many obstacles and nowadays is readily applicable for structural analysis of all types of compounds and materials. Being less straightforward than single crystal diffraction, it requires significant users’ input and consequently, implementation of standardized tools to assess the accuracy of crystal structures. This article discusses potential errors in crystal structure solution and refinement of small‐molecule structures obtained from PXRD data. Moreover, it proposes how accuracy of these structures can be improved by using high‐quality PXRD data, complementary external analytical techniques, knowledge stored in crystal structure databases, as well as an approach to search the parameter space to avoid local minima in testing different sets of geometry restraints.

Last Word

At issue is the suppression of news in South Africa. Ken Owen, editor of Busi- ness Day, states in this article: "There is a vast difference between a system that limits expression by law, subject to the judgement of the courts, and one which seeks to impose a vaguely de- fined set of restraints that go beyond the law". He argues that South Africa has been moving from the former system to the latter, casting law aside. But calls for censorship of the news, couched as "greater responsibility", or "better judgement", even "patriotism", ema- nate from all quarters. All depending on whose ox is gored, writes Mr Owen.

Characterization of protein complexes in extracellular vesicles by intact extracellular vesicle crosslinking mass spectrometry (iEVXL)

Extracellular vesicles (EVs) are blood‐borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein‐protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient‐cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs. By using a partially membrane‐permeable disuccinimidyl suberate crosslinker, proteins on the EV outer‐surface and inside EVs can be immobilized together with their interacting partners. This not only provides covalent stabilization of protein complexes before extraction from the membrane‐enclosed environment, but also generates a set of crosslinking distance restraints that can be used for structural modelling and comparative screening of changes in EV protein assemblies. Here we demonstrate iEVXL as a powerful approach to reveal high‐resolution information, about protein determinants that govern EV docking and signalling, and as a crucial aid in modelling docking interactions.

Effects of Surface-Parallel Edge Restraints and Inter-laminar Shear on the Responses of Doubly Curved General Cross-Ply Panels

This study presents first a relatively lesser studied topic of the role played by surface-parallel restraints in determining the response of simply supported thick to thin doubly curved cross-ply panels of rectangular plan-form, modeled using a third order shear deformation theory, quantified by way of the difference between full and absent surface-parallel edge restraints. Mathematically speaking, this corresponds to the difference between complementary solutions to mixed boundary-value problems, resulting from two extreme sets of surface-parallel restraints.

Minimal Increments of Hydrophobic Collapse within the N-Terminus of the Neuropeptide Galanin.

The neuropeptide galanin has a 35-year history as an intriguing target in drug design owing to its implication as a potential anticonvulsant and neuronal trophic factor among many other therapeutically interesting functions including analgesia and mood alteration. In this study, we report the structural characterization of three synthetic fragments of the galanin N-terminus in buffered aqueous solution: hGal(2-12)KK, hGal(1-12)KK, and hGal(1-17)KK. High-field two-dimensional 1H-1H nuclear magnetic resonance (NMR) data were acquired for these fragments and used to derive distance restraints. We further utilized modified hydrogen bonding and dihedral restraints to reflect chemical shift patterns in the data, which revealed the signature of a weakly folded helix. Together, these sets of restraints were used to generate NMR structures of all three fragments, which depict a core of hydrophobic residues that cluster together regardless of the presence of a helical structure, and correspond to residues in the N-terminus of galanin that have been previously shown to be critical for binding its receptors. The helical structure only appears following the inclusion of Gly(1) in the sequence, and at longer sequence lengths, unlike many other peptides, the helix does not propagate. Rather, a few turns of poorly ordered helix appear to be a secondary consequence of clusters of hydrophobic sidechains that are conserved across all of the peptides in this study; the helices themselves appear ordered as a consequence of this clustering, and these clusters compare directly to those observed recently to make contacts between galanin and two of its receptor subtypes. Collapsed hydrophobic residues therefore organize and compose the functional core of human galanin and raise interesting questions about the nature of the conformational order in ligands that bind cell surface receptors.

Standard Binding Free Energy and Membrane Desorption Mechanism for a Phospholipase C.

Peripheral membrane proteins (PMPs) bind temporarily to cellular membranes and play important roles in signaling, lipid metabolism, and membrane trafficking. Obtaining accurate membrane-PMP affinities using experimental techniques is more challenging than for protein-ligand affinities in an aqueous solution. At the theoretical level, calculation of the standard protein-membrane binding free energy using molecular dynamics simulations remains a daunting challenge owing to the size of the biological objects at play, the slow lipid diffusion, and the large variation in configurational entropy that accompanies the binding process. To overcome these challenges, we used a computational framework relying on a series of potential-of-mean-force (PMF) calculations including a set of geometrical restraints on collective variables. This methodology allowed us to determine the standard binding free energy of a PMP to a phospholipid bilayer using an all-atom force field. Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) was chosen due to its importance as a virulence factor and owing to the host of experimental affinity data available. We computed a standard binding free energy of -8.2 ± 1.4 kcal/mol in reasonable agreement with the reported experimental values (-6.6 ± 0.2 kcal/mol). In light of the 2.3-μs separation PMF calculation, we investigated the mechanism whereby BtPI-PLC disengages from interactions with the lipid bilayer during separation. We describe how a short amphipathic helix engages in transitory interactions to ease the passage of its hydrophobes through the interfacial region upon desorption from the bilayer.

Arginine off-kilter: guanidinium is not as planar as restraints denote.

Crystallographic refinement of macromolecular structures relies on stereochemical restraints to mitigate the typically poor data-to-parameter ratio. For proteins, each amino acid has a unique set of geometry restraints which represent stereochemical information such as bond lengths, valence angles, torsion angles, dihedrals and planes. It has been shown that the geometry in refined structures can differ significantly from that present in libraries; for example, it was recently reported that the guanidinium moiety in arginine is not symmetric. In this work, the asymmetry of the Nϵ-Cζ-Nη1 and Nϵ-Cζ-Nη2 valence angles in the guanidinium moiety is confirmed. In addition, it was found that the Cδ atom can deviate significantly (more than 20°) from the guanidinium plane. This requires the relaxation of the planar restraint for the Cδ atom, as it otherwise causes the other atoms in the group to compensate by distorting the guanidinium core plane. A new set of restraints for the arginine side chain have therefore been formulated, and are available in the software package Phenix, that take into account the asymmetry of the group and the planar deviation of the Cδ atom. This is an example of the need to regularly revisit the geometric restraint libraries used in macromolecular refinement so that they reflect the best knowledge of the structural chemistry of their components available at the time.

PRE-driven protein NMR structures: an alternative approach in highly paramagnetic systems.

Metalloproteins play key roles across biology, and knowledge of their structure is essential to understand their physiological role. For those metalloproteins containing paramagnetic states, the enhanced relaxation caused by the unpaired electrons often makes signal detection unfeasible near the metal center, precluding adequate structural characterization right where it is more biochemically relevant. Here, we report a protein structure determination by NMR where two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs), are used separately and eventually together. The protein PioC from Rhodopseudomonas palustris TIE-1 is a High Potential Iron-Sulfur Protein (HiPIP) where the [4Fe-4S] cluster is paramagnetic in both oxidation states at room temperature providing the source of PREs used as alternative distance restraints. Comparison of the family of structures obtained using NOEs only, PREs only, and the combination of both reveals that the pairwise root-mean-square deviation (RMSD) between them is similar and comparable with the precision within each family. This demonstrates that, under favorable conditions in terms of protein size and paramagnetic effects, PREs can efficiently complement and eventually replace NOEs for the structural characterization of small paramagnetic metalloproteins and de novo-designed metalloproteins by NMR. DATABASES: The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487).

Investigating aged binder mobilization and performance of RAP mixtures for surface courses

Pavement engineering practice still deals with a set of restraints regarding the use of RAP, mainly in the case of bituminous surface courses. The main objectives of this paper are to assess: (a) the influence of the rejuvenator and the degree of aged binder mobilization on the homogeneity of bituminous mixtures; and (b) the performance and durability of bituminous mixtures for surface courses with 25% RAP. The laboratory investigation studied an AC 14 surf 35/50 considering two mixture compositions: a reference mixture with virgin constituents; and a mixture containing RAP and a rejuvenator agent. The study was focused on the mechanical and functional behaviours, as well as on the short- and long-term ageing performance of mixtures. An experimental protocol for RAP fractioning and rejuvenator addition was established to achieve a better control of the final mixture quality. The aged binder mobilization was more efficient when RAP was heated. The RAP mixture revealed a good performance in terms of permanent deformation, water sensitivity, stiffness, and fatigue. The incorporation of fractionated RAP allowed to have a mixture with good surface characteristics. Aged RAP mixture performance envisioning good long-term behaviour. Regarding the promotion of the circular economy, this investigation clearly demonstrated that mixtures with a 25% RAP incorporation can be currently used in surface courses.

Quantum refinement with multiple conformations: application to the P-cluster in nitrogenase.

X-ray crystallography is the main source of atomistic information on the structure of proteins. Normal crystal structures are obtained as a compromise between the X-ray scattering data and a set of empirical restraints that ensure chemically reasonable bond lengths and angles. However, such restraints are not always available or accurate for nonstandard parts of the structure, for example substrates, inhibitors and metal sites. The method of quantum refinement, in which these empirical restraints are replaced by quantum-mechanical (QM) calculations, has previously been suggested for small but interesting parts of the protein. Here, this approach is extended to allow for multiple conformations in the QM region by performing separate QM calculations for each conformation. This approach is shown to work properly and leads to improved structures in terms ofelectron-density maps and real-space difference density Z-scores. It is also shown that the quality of the structures can be gauged using QM strain energies. The approach, called ComQumX-2QM, is applied to the P-cluster in two different crystal structures of the enzyme nitrogenase, i.e. an Fe8S7Cys6 cluster, used for electron transfer. One structure is at a very high resolution (1.0 Å) and shows a mixture of two different oxidation states, the fully reduced PN state (Fe82+, 20%) and the doubly oxidized P2+ state (80%). In the original crystal structure the coordinates differed for only two iron ions, but here it is shown that the two states also show differences in other atoms of up to 0.7 Å. The second structure is at a more modest resolution, 2.1 Å, and was originally suggested to show only the one-electron oxidized state, P1+. Here, it is shown that it is rather a 50/50% mixture of the P1+ and P2+ states and that many of the Fe-Fe and Fe-S distances in the original structure were quite inaccurate (by up to 0.8 Å). This shows that the new ComQumX-2QM approach can be used to sort out what is actually seen in crystal structures with dual conformations and to give locally improved coordinates.

Closed-Form Solutions for Initial Thermal Postbuckling Response of Axially Restrained Columns

AbstractClosed-form equations for the initial thermal postbuckling response of axially restrained elastic columns with six well-known sets of rotational and lateral end restraints are presented. Th...

Validation of protein backbone structures calculated from NMR angular restraints using Rosetta.

Multidimensional solid-state NMR spectra of oriented membrane proteins can be used to infer the backbone torsion angles and hence the overall protein fold by measuring dipolar couplings and chemical shift anisotropies, which depend on the orientation of each peptide plane with respect to the external magnetic field. However, multiple peptide plane orientations can be consistent with a given set of angular restraints. This ambiguity is further exacerbated by experimental uncertainty in obtaining and interpretingsuch restraints. The previously developed algorithms for structure calculations using angular restraints typically involve a sequential walkthrough along the backbone to find the torsion angles between the consecutive peptide plane orientations that are consistent with the experimental data. This method is sensitive to experimental uncertainty in interpreting the peak positions of as low as ± 10Hz, often yielding high structural RMSDs for the calculated structures. Here we present a significantly improved version of the algorithm which includes the fitting of several peptide planes at once in order to prevent propagation of error along the backbone. In addition, a protocol has been devised for filtering the structural solutions using Rosetta scoring functions in order to find the structures that both fit the spectrum and satisfy bioinformatics restraints. The robustness of the new algorithm has been tested using synthetic angular restraints generated from the known structures for two proteins: a soluble protein 2gb1 (56 residues), chosen for its diverse secondary structure elements, i.e. an alpha-helix and two beta-sheets, and a membrane protein 4a2n, from which the first two transmembrane helices (having a total of 64 residues) have been used. Extensive simulations have been performed by varying the number of fitted planes, experimental error, and the number of NMR dimensions. It has been found that simultaneously fitting two peptide planes always shifted the distribution of the calculated structures toward lower structural RMSD values as compared to fitting a single torsion-angle pair. For each protein, irrespective of the simulation parameters, Rosetta was able to distinguish the most plausible structures, often having structural RMSDs lower than 2Å with respect to the original structure. This study establishes a framework for de-novo protein structure prediction using a combination of solid-state NMR angular restraints and bioinformatics.

Parametric analysis on external dissipative link system for seismic protection of low rise r.c. buildings

The seismic rehabilitation of existing r.c. and masonry buildings is a topical issue in Civil Engineering. A useful technique consists in the introduction of external bracing system. This kind of intervention can be improved throughout the introduction of dissipative links, here investigated with the help of some examples.. The links are made of common steel profiles: length, geometry and its disposition are considered as design parameters. A general procedure of link-bracing optimization is proposed applying a set of identical external restraints . The adopted dissipative links consent a cheaper and effective design both in terms of ULS than in DLS: the damages are addressed in a small area permitting an easy replacement of the links in case of earthquake.

Challenge in Management of Aggressive Clients in Psychiatric Setting: Development of Four-Point Physical Restraints

Introduction: The use of physical restraints is a very common procedure in psychiatric setting from the beginning of psychiatry as a branch of medicine. Any measure that keep someone under control is called a restraint. Four-point physical restraints are required for managing clients with mental illness. Material and Method: The objective of the project was to develop a safe and secure soft limb restraints for aggressive clients. A set of soft limb restraints (two each for upper and lower limbs) was developed along with user guidelines. High quality narrow woven fabric material was selected for preparing the restraints. The restraints were tested in 5 psychiatric hospitals. A 12 item checklist was developed and validated for assessing the user friendliness of the restraints. Necessary modifications were made based on the feedback given by the nurses who tested it. Results: All the nurses (100%) reported that the developed restraints were easy to apply, easy to remove and met the safety of the clients. Conclusion: The restraints developed was found to be more safe and operable as compared to other commercially available restraints.

Iron-sulfur clusters have no right angles.

Accurate geometric restraints are vital in the automation of macromolecular crystallographic structure refinement. A set of restraints for the Fe4S4 cubane-type cluster was created using the Cambridge Structural Database (CSD) and high-resolution structures from the Protein Data Bank. Geometries from each source were compared and pairs of refinements were performed to validate these new restraints. In addition to the restraints internal to the cluster, the CSD was mined to generate bond and angle restraints to be applied to the most common linking motif for Fe4S4: coordination of the four Fe atoms to the side-chain sulfurs of four cysteine residues. Furthermore, computational tools were developed to assist researchers when refining Fe4S4-containing proteins.

The productive-unproductive puzzle in Adam Smith: what have we missed?

This paper returns to Adam Smith's much maligned distinction of productive versus unproductive labour to flesh out a dimension of capital productivity that has been missed by the modern theory of production and welfare. The puzzle lies in Smith's use of a binary measurement scale to suggest opposing productivity, and household welfare outcomes obtained with a durable versus non-durable good. A durable good generates a permanent fund of labour savings and service spillovers over time. This dimension of productivity exists separate from, and beyond, any marginal productivity attributed to capital or labour services within the neoclassical production function. And, it forms the basis of improvements in household welfare that Smith described in the Wealth of Nations – in terms of continual net increases in the consumption-production possibility frontier enjoyed by the household, as a result of service spillovers obtained with a durable good over time. In contrast, no such spillovers are obtained with a non-durable good. A preference-bias for non-durable goods, instead, proves to be welfare-reducing – by having households under-invest and/or fail in maintaining the accumulated stock of durable-goods-as-capital. Both lead to a loss of production-consumption possibilities available within the household economy. An exploration of Smith's concern with ‘unproductive labour‘ brings back into focus the broad set of behavioural traits and ethicallegal restraints that underlie economic progress and have been missed by neoclassical theory.

Digital Constitutionalism: Using the Rule of Law to Evaluate the Legitimacy of Governance by Platforms

Platforms govern users, and the way that platforms govern matters. In this paper, I propose that the legitimacy of governance of users by platforms should be evaluated against the values of the rule of law. In particular, I suggest that we should care deeply about the extent to which private governance is consensual; transparent; equally applied and relatively stable; fairly enforced; and respects substantive human rights. These are the core values of good governance, but are alien to the systems of contract law that currently underpin relationships between platforms and their users. Through an analysis of the contractual terms of service of 15 major social media platforms, I show how these values can be applied to evaluate governance, and how poorly platforms perform on these criteria. The values of the rule of law provide a language to name and work through contested concerns about the relationship between platforms and their users. This is a necessary precondition to an increasingly urgent task. The law of contract does not address these governance concerns, and the concept of constitutional rights does not extend to governance by private actors. Finding a way to apply these values to articulate a set of desirable restraints on the exercise of power in the digital age is the key challenge and opportunity of the project of digital constitutionalism.