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Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard β-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a "preemptive holdase" mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function.

Due to its symmetric structure and abundance of carboxyl groups, mellitic acid (MA–benzenehexacarboxylic acid) has an uncommon capacity to form highly ordered molecular networks. Dissolved in water, MA dissociates to yield various mellitate anions with pronounced tendencies to form complexes with cations including protonated amines. Deprotonation of MA at physiological pH produces anions with high charge densities (MA5- and MA6-) whose influence on co-dissolved proteins has not been thoroughly studied. As electrostatic attraction between highly symmetric MA6- anions and positively charged low-symmetry globular proteins could lead to interesting self-assembly patterns we have chosen hen egg white lysozyme (HEWL), a basic stably folded globular protein as a cationic partner for mellitate anions to form such hypothetical nanostructures. Indeed, mixing of neutral HEWL and MA solutions does result in precipitation of electrostatic complexes with the stoichiometry dependent on pH. We have studied the self-assembly of HEWL-MA structures using vibrational spectroscopy (infrared absorption and Raman scattering), circular dichroism (CD), atomic force microscopy (AFM). Possible HEWL-MA6- molecular docking scenarios were analyzed using computational tools. Our results indicate that even at equimolar ratios (in respect to HEWL), MA5- and MA6- anions are capable of inducing misfolding and aggregation of the protein upon mild heating which results in non-native intermolecular beta-sheet appearing in the amide I’ region of the corresponding infrared spectra. The association process leads to aggregates with compacted morphologies entrapping mellitate anions. The capacity of extremely diluted mellitate anions (i.e. at sub-millimolar concentration range) to trigger aggregation of proteins is discussed in the context of mechanisms of misfolding.

Is it possible to compare the physicochemical properties of a wild-type protein and its mutant form under the same conditions? Provided the mutation has destabilized the protein, it may be more correct to compare the mutant protein under native conditions to the wild-type protein destabilized with a small amount of the denaturant. In general, is it appropriate to compare the properties of proteins destabilized by different treatments: mutations, pH, temperature, and denaturants like urea? These issues have compelled us to search for methods and ways of presentation of experimental results that would allow a comparison of mutant forms of proteins under different conditions and lead to conclusions on the effect of mutations on the protein folding/unfolding pathway. We have studied equilibrium unfolding of wild-type bovine carbonic anhydrase II (BCA II) and its six mutant forms using different urea concentrations. BCA II has been already studied in detail and is a good model object for validating new techniques. In this case, time-resolved fluorescence spectroscopy was chosen as the basic research method. The main features of this experimental method allowed us to compare different stages of unfolding of studied proteins and prove experimentally that a single substitution of the amino acid in three mutant forms of BCA II affected the native state of the protein but did not change its unfolding pathway. On the contrary, the inserted disulfide bridge in three other mutant forms of BCA II affected the protein unfolding pathway. An important result of this research is that we have validated the new approach allowing investigation of the effect of mutations on the folding of globular proteins, because in this way it is possible to compare proteins in the same structural states rather than under identical conditions.

Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

Changes in the tertiary structure of proteins and the resultant fibrillary aggregation could result in fatal heredity diseases, such as lysozyme systemic amyloidosis. Human lysozyme is a globular protein with antimicrobial properties with tendencies to fibrillate and hence is known as a fibril-forming protein. Therefore, its behavior under different ambient conditions is of great importance. In this study, we conducted two 500000 ps molecular dynamics (MD) simulations of human lysozyme in sodium dodecyl sulfate (SDS) at two ambient temperatures. To achieve comparative results, we also performed two 500000 ps human lysozyme MD simulations in pure water as controls. The aim of this study was to provide further molecular insight into all interactions in the lysozyme-SDS complexes and to provide a perspective on the ability of human lysozyme to form amyloid fibrils in the presence of SDS surfactant molecules. SDS, which is an anionic detergent, contains a hydrophobic tail with 12 carbon atoms and a negatively charged head group. The SDS surfactant is known to be a stabilizer for helical structures above the critical micelle concentration (CMC) [1]. During the 500000 ps MD simulations, the helical structures were maintained by the SDS surfactant above its CMC at 300 K, while at 370 K, human lysozyme lost most of its helices and gained β-sheets. Therefore, we suggest that future studies investigate the β-amyloid formation of human lysozyme at SDS concentrations above the CMC and at high temperatures.

Molecular dynamics simulations are used to explore the effects of chaperonin-like cages on knotted proteins with very low sequence similarity, different depths of a knot but with a similar fold, and the same type of topology. The investigated proteins are VirC2, DndE and MJ0366 with two depths of a knot. A comprehensive picture how encapsulation influences folding rates is provided based on the analysis of different cage sizes and temperature conditions. Neither of these two effects with regard to knotted proteins has been studied by means of molecular dynamics simulations with coarse-grained structure-based models before. We show that encapsulation in a chaperonin is sufficient to self-tie and untie small knotted proteins (VirC2, DndE), for which the equilibrium process is not accessible in the bulk solvent. Furthermore, we find that encapsulation reduces backtracking that arises from the destabilisation of nucleation sites, smoothing the free energy landscape. However, this effect can also be coupled with temperature rise. Encapsulation facilitates knotting at the early stage of folding and can enhance an alternative folding route. Comparison to unknotted proteins with the same fold shows directly how encapsulation influences the free energy landscape. In addition, we find that as the size of the cage decreases, folding times increase almost exponentially in a certain range of cage sizes, in accordance with confinement theory and experimental data for unknotted proteins.

Protein O-GlcNAcylation (O-GlcNAc) is an essential post-translational modification (PTM) in higher eukaryotes. The O-linked β-N-acetylglucosamine transferase (OGT), targets specific Serines and Threonines (S/T) in intracellular proteins. However, unlike phosphorylation, fewer than 25% of known O-GlcNAc sites match a clear sequence pattern. Accordingly, the three-dimensional structures of O-GlcNAc sites were characterised to investigate the role of structure in molecular recognition. From 1,584 O-GlcNAc sites in 620 proteins, 143 were mapped to protein structures determined by X-ray crystallography. The modified S/T were 1.7 times more likely to be annotated in the REM465 field which defines missing residues in a protein structure, while 7 O-GlcNAc sites were solvent inaccessible and unlikely to be targeted by OGT. 132 sites with complete backbone atoms clustered into 10 groups, but these were indistinguishable from clusters from unmodified S/T. This suggests there is no prevalent three-dimensional motif for OGT recognition. Predicted features from the 620 proteins were compared to unmodified S/T in O-GlcNAcylated proteins and globular proteins. The Jpred4 predicted secondary structure shows that modified S/T were more likely to be coils. 5/6 methods to predict intrinsic disorder indicated O-GlcNAcylated S/T to be significantly more disordered than unmodified S/T. Although the analysis did not find a pattern in the site three-dimensional structure, it revealed the residues around the modification site are likely to be disordered and suggests a potential role of secondary structure elements in OGT site recognition.

In living systems proteins are typically found in crowded environments where their effective interactions strongly depend on the surrounding medium. Yet, their association and dissociation needs to be robustly controlled in order to enable biological function. Uncontrolled protein aggregation often causes disease. For instance, cataract is caused by the clustering of lens proteins, i.e., crystallins, resulting in enhanced light scattering and impaired vision or blindness. To investigate the molecular origins of cataract formation and to design efficient treatments, a better understanding of crystallin association in macromolecular crowded environment is needed. Here we present a theoretical study of simple coarse grained colloidal models to characterize the general features of how the association equilibrium of proteins depends on the magnitude of intermolecular attraction. By comparing the analytic results to the available experimental data on the osmotic pressure in crystallin solutions, we identify the effective parameters regimes applicable to crystallins. Moreover, the combination of two models allows us to predict that the number of binding sites on crystallin is small, i.e. one to three per protein, which is different from previous estimates. We further observe that the crowding factor is sensitive to the size asymmetry between the reactants and crowding agents, the shape of the protein clusters, and to small variations of intermolecular attraction. Our work may provide general guidelines on how to steer the protein interactions in order to control their association.

Calculation of the free energy of protein folding and delineation of its pre-organization are of foremost importance for understanding, predicting and designing biological macromolecules. Here, we introduce an energy smoothing variant of parallel tempering replica exchange Monte Carlo (REMS) that allows for efficient configurational sampling of flexible solutes under the conditions of molecular hydration. Its usage to calculate the thermal stability of a model globular protein, Trp cage TC5b, achieves excellent agreement with experimental measurements. We find that the stability of TC5b is attained through the coupled formation of local and non-local interactions. Remarkably, many of these structures persist at high temperature, concomitant with the origin of native-like configurations and mesostates in an otherwise macroscopically disordered unfolded state. Graph manifold learning reveals that the conversion of these mesostates to the native state is structurally heterogeneous, and that the cooperativity of their formation is encoded largely by the unfolded state ensemble. In all, these studies establish the extent of thermodynamic and structural pre-organization of folding of this model globular protein, and achieve the calculation of macromolecular stability ab initio, as required for ab initio structure prediction, genome annotation, and drug design.

The accurate characterization of proteins in both their native and denatured states is essential to effectively understand protein function, folding and stability. As a proof of concept, a micro rheological method is applied, based on the characterization of thermal fluctuations of a micro cantilever immersed in a bovine serum albumin solution, to assess changes in the viscosity associated with modifications in the protein's structure under the denaturant effect of urea. Through modeling the power spectrum density of the cantilever's fluctuations over a broad frequency band, it is possible to implement a fitting procedure to accurately determine the viscosity of the fluid, even at low volumes. Increases in viscosity during the denaturant process are identified using the assumption that the protein is a hard sphere, with a hydrodynamic radius that increases during unfolding. This is modeled accordingly through the Einstein-Batchelor formula. The Einstein-Batchelor formula estimates are verified through dynamic light scattering, which measures the hydrodynamic radius of proteins. Thus, this methodology is proven to be suitable for the study of protein folding in samples of small size at vanishing shear stresses.

Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)—like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners.

Hierarchical organization of free energy landscape (FEL) for native globular proteins has been widely accepted by the biophysics community. However, FEL of native proteins is usually projected onto one or a few dimensions. Here we generated collectively 0.2 milli-second molecular dynamics simulation trajectories in explicit solvent for hen egg white lysozyme (HEWL), and carried out detailed conformational analysis based on backbone torsional degrees of freedom (DOF). Our results demonstrated that at micro-second and coarser temporal resolutions, FEL of HEWL exhibits hub-like topology with crystal structures occupying the dominant structural ensemble that serves as the hub of conformational transitions. However, at 100 ns and finer temporal resolutions, conformational substates of HEWL exhibit network-like topology, crystal structures are associated with kinetic traps that are important but not dominant ensembles. Backbone torsional state transitions on time scales ranging from nanoseconds to beyond microseconds were found to be associated with various types of molecular interactions. Even at nanoseconds temporal resolution, the number of conformational substates that are of statistical significance is quite limited. These observations suggest that detailed analysis of conformational substates at multiple temporal resolutions is both important and feasible. Transition state ensembles among various conformational substates at microsecond temporal resolution were observed to be considerably disordered. Life times of these transition state ensembles are found to be nearly independent of the time scales of the participating torsional DOFs.

Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.

Horses are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein. The amino acid sequence of latherin, determined from cDNA analysis, is highly conserved across four geographically dispersed equid species (horse, zebra, onager, ass), and is similar to a family of proteins only found previously in the oral cavity and associated tissues of mammals. Latherin produces a significant reduction in water surface tension at low concentrations (< or = 1 mg ml(-1)), and therefore probably acts as a wetting agent to facilitate evaporative cooling through a waterproofed pelt. Neutron reflection experiments indicate that this detergent-like activity is associated with the formation of a dense protein layer, about 10 A thick, at the air-water interface. However, biophysical characterization (circular dichroism, differential scanning calorimetry) in solution shows that latherin behaves like a typical globular protein, although with unusual intrinsic fluorescence characteristics, suggesting that significant conformational change or unfolding of the protein is required for assembly of the air-water interfacial layer. RT-PCR screening revealed latherin transcripts in horse skin and salivary gland but in no other tissues. Recombinant latherin produced in bacteria was also found to be the target of IgE antibody from horse-allergic subjects. Equids therefore may have adapted an oral/salivary mucosal protein for two purposes peculiar to their lifestyle, namely their need for rapid and efficient heat dissipation and their specialisation for masticating and processing large quantities of dry food material.

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5-1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).

Monoclonal antibodies represent the most important group of protein-based biopharmaceuticals. During formulation, manufacturing, or storage, antibodies may suffer post-translational modifications altering their physical and chemical properties. Such induced conformational changes may lead to the formation of aggregates, which can not only reduce their efficiency but also be immunogenic. Therefore, it is essential to monitor the amount of size variants to ensure consistency and quality of pharmaceutical antibodies. In many cases, antibodies are formulated at very high concentrations > 50 g/L, mostly along with high amounts of sugar-based excipients. As a consequence, all routine aggregation analysis methods, such as size-exclusion chromatography, cannot monitor the size distribution at those original conditions, but only after dilution and usually under completely different solvent conditions. In contrast, sedimentation velocity (SV) allows to analyze samples directly in the product formulation, both with limited sample-matrix interactions and minimal dilution. One prerequisite for the analysis of highly concentrated samples is the detection of steep concentration gradients with sufficient resolution: Commercially available ultracentrifuges are not able to resolve such steep interference profiles. With the development of our Advanced Interference Detection Array (AIDA), it has become possible to register interferograms of solutions as highly concentrated as 150 g/L. The other major difficulty encountered at high protein concentrations is the pronounced non-ideal sedimentation behavior resulting from repulsive intermolecular interactions, for which a comprehensive theoretical modelling has not yet been achieved. Here, we report the first SV analysis of highly concentrated antibodies up to 147 g/L employing the unique AIDA ultracentrifuge. By developing a consistent experimental design and data fit approach, we were able to provide a reliable estimation of the minimum content of soluble aggregates in the original formulations of two antibodies. Limitations of the procedure are discussed.

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function.

Macromolecular crowding has been shown to have an exacerbating effect on the aggregation propensity of amyloidogenic proteins; while having an inhibitory effect on the non-amyloidogenic proteins. However, the results concerning aggregation propensity of non-amyloidogenic proteins have not been convincing due to the contrasting effect on holo-LA, which despite being a non-amyloidogenic protein was observed to aggregate under crowded conditions. In the present study, we have extensively characterized the crowding-induced holo-LA aggregates and investigated the possible mechanism responsible for the aggregation process. We discovered that macromolecular crowding reduces the calcium binding affinity of holo-LA resulting in the formation of apo-LA (the calcium-depleted form of holo-LA) leading to aggregate formation. Another finding is that calcium acts as a chaperone capable of inhibiting and dissociating crowding-induced holo-LA aggregates. The study has a direct implication to Alzheimer Disease as the results invoke a new mechanism to prevent Aβ fibrillation.

Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.

Proteins accomplish their physiological functions with remarkably organized dynamic transitions among a hierarchical network of conformational substates. Despite the essential contribution of water molecules in shaping functionally important protein dynamics, their exact role is still controversial. Water molecules were reported either as mediators that facilitate or as masters that slave protein dynamics. Since dynamic behaviour of a given protein is ultimately determined by the underlying energy landscape, we systematically analysed protein self energies and protein-water interaction energies obtained from extensive molecular dynamics simulation trajectories of barstar. We found that protein-water interaction energy plays the dominant role when compared with protein self energy, and these two energy terms on average have negative correlation that increases with increasingly longer time scales ranging from 10 femtoseconds to 100 nanoseconds. Water molecules effectively roughen potential energy surface of proteins in the majority part of observed conformational space and smooth in the remaining part. These findings support a scenario wherein water on average slave protein conformational dynamics but facilitate a fraction of transitions among different conformational substates, and reconcile the controversy on the facilitating and slaving roles of water molecules in protein conformational dynamics.

The control of protein aggregation is an important requirement in the development of bio-pharmaceutical formulations. Here a simple protein model is proposed that was used in molecular dynamics simulations to obtain a quantitative assessment of the relative contributions of proteins’ net-charges, dipole-moments, and the size of hydrophobic or charged surface patches to their colloidal interactions. The results demonstrate that the strength of these interactions correlate with net-charge and dipole moment. Variation of both these descriptors within ranges typical for globular proteins have a comparable effect. By comparison no clear trends can be observed upon varying the size of hydrophobic or charged patches while keeping the other parameters constant. The results are discussed in the context of experimental literature data on protein aggregation. They provide a clear guide line for the development of improved algorithms for the prediction of aggregation propensities.

International audience; We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution.

Three-dimensional protein structures usually contain regions of local order, called secondary structure, such as $\alpha$-helices and $\beta$-sheets. Secondary structure is characterized by the local rotational state of the protein backbone, quantified by two dihedral angles called $\phi$ and $\psi$. Particular types of secondary structure can generally be described by a single (diffuse) location on a two-dimensional plot drawn in the space of the angles $\phi$ and $\psi$, called a Ramachandran plot. By contrast, a recently-discovered nanomaterial made from peptoids, structural isomers of peptides, displays a secondary-structure motif corresponding to two regions on the Ramachandran plot [Mannige et al., Nature 526, 415 (2015)]. In order to describe such `higher-order' secondary structure in a compact way we introduce here a means of describing regions on the Ramachandran plot in terms of a single Ramachandran number, ${\mathcal{R}}$, which is a structurally meaningful combination of $\phi$ and $\psi$. We show that the potential applications of ${\mathcal{R}}$ are numerous: it can be used to describe the geometric content of protein structures, and can be used to draw diagrams that reveal, at a glance, the frequency of occurrence of regular secondary structures and disordered regions in large protein datasets. We propose that ${\mathcal{R}}$ might be used as an order parameter for protein geometry for a wide range of applications.

The purpose of this work was to construct a consensus prediction algorithm of ‘aggregation-prone’ peptides in globular proteins, combining existing tools. This allows comparison of the different algorithms and the production of more objective and accurate results. Eleven (11) individual methods are combined and produce AMYLPRED2, a publicly, freely available web tool to academic users (http://biophysics.biol.uoa.gr/AMYLPRED2), for the consensus prediction of amyloidogenic determinants/‘aggregation-prone’ peptides in proteins, from sequence alone. The performance of AMYLPRED2 indicates that it functions better than individual aggregation-prediction algorithms, as perhaps expected. AMYLPRED2 is a useful tool for identifying amyloid-forming regions in proteins that are associated with several conformational diseases, called amyloidoses, such as Altzheimer's, Parkinson's, prion diseases and type II diabetes. It may also be useful for understanding the properties of protein folding and misfolding and for helping to the control of protein aggregation/solubility in biotechnology (recombinant proteins forming bacterial inclusion bodies) and biotherapeutics (monoclonal antibodies and biopharmaceutical proteins).

Histone proteins are not only important due to their vital role in cellular processes such as DNA compaction, replication and repair but also show intriguing structural properties that might be exploited for bioengineering purposes such as the development of nano-materials. Based on their biological and technological implications, it is interesting to investigate the structural properties of proteins as a function of temperature. In this work, we study the spatial response dynamics of the histone H2AX, consisting of 143 residues, by a coarse-grained bond fluctuating model for a broad range of normalized temperatures. A knowledge-based interaction matrix is used as input for the residue-residue Lennard-Jones potential.We find a variety of equilibrium structures including global globular configurations at low normalized temperature (T* = 0.014), combination of segmental globules and elongated chains (T* = 0.016,0.017), predominantly elongated chains (T* = 0.019,0.020), as well as universal SAW conformations at high normalized temperature (T* ≥ 0.023). The radius of gyration of the protein exhibits a non-monotonic temperature dependence with a maximum at a characteristic temperature (T(c)* = 0.019) where a crossover occurs from a positive (stretching at T* ≤ T(c)*) to negative (contraction at T* ≥ T(c)*) thermal response on increasing T*.

Amyloid fibrillar aggregates of polypeptides are associated with many neurodegenerative diseases. Short peptide segments in protein sequences may trigger aggregation. Identifying these stretches and examining their behavior in longer protein segments is critical for understanding these diseases and obtaining potential therapies. In this study, we combined machine learning and structure-based energy evaluation to examine and predict amyloidogenic segments. Our feature selection method discovered that windows consisting of long amino acid segments of ∼30 residues, instead of the commonly used short hexapeptides, provided the highest accuracy. Weighted contributions of an amino acid at each position in a 27 residue window revealed three cooperative regions of short stretch, resemble the β-strand-turn-β-strand motif in A-βpeptide amyloid and β-solenoid structure of HET-s(218–289) prion (C). Using an in-house energy evaluation algorithm, the interaction energy between two short stretches in long segment is computed and incorporated as an additional feature. The algorithm successfully predicted and classified amyloid segments with an overall accuracy of 75%. Our study revealed that genome-wide amyloid segments are not only dependent on short high propensity stretches, but also on nearby residues.

β-barrel membrane proteins play an important role in controlling the exchange and transport of ions and organic molecules across bacterial and mitochondrial outer membranes. They are also major regulators of apoptosis and are important determinants of bacterial virulence. In contrast to β-helical membrane proteins, their evolutionary pattern of residue substitutions has not been quantified, and there are no scoring matrices appropriate for their detection through sequence alignment. Using a Bayesian Monte Carlo estimator, we have calculated the instantaneous substitution rates of transmembrane domains of bacterial β-barrel membrane proteins. The scoring matrices constructed from the estimated rates, called bbTM for β-barrel Transmembrane Matrices, improve significantly the sensitivity in detecting homologs of β-barrel membrane proteins, while avoiding erroneous selection of both soluble proteins and other membrane proteins of similar composition. The estimated evolutionary patterns are general and can detect β-barrel membrane proteins very remote from those used for substitution rate estimation. Furthermore, despite the separation of 2-3 billion years since the proto-mitochondrion entered the proto-eukaryotic cell, mitochondria outer membrane proteins in eukaryotes can also be detected accurately using these scoring matrices derived from bacteria. This is consistent with the suggestion that there is no eukaryote-specific signals for translocation. With these matrices, remote homologs of β-barrel membrane proteins with known structures can be reliably detected at genome scale, allowing construction of high quality structural models of their transmembrane domains, at the rate of 131 structures per template protein. The scoring matrices will be useful for identification, classification, and functional inference of membrane proteins from genome and metagenome sequencing projects. The estimated substitution pattern will also help to identify key elements important for the structural and functional integrity of β-barrel membrane proteins, and will aid in the design of mutagenesis studies.

Integral membrane proteins constitute 25-30% of genomes and play crucial roles in many biological processes. However, less than 1% of membrane protein structures are in the Protein Data Bank. In this context, it is important to develop reliable computational methods for predicting the structures of membrane proteins. Here, we present the first application of random forest (RF) for residue-residue contact prediction in transmembrane proteins, which we term as TMhhcp. Rigorous cross-validation tests indicate that the built RF models provide a more favorable prediction performance compared with two state-of-the-art methods, i.e., TMHcon and MEMPACK. Using a strict leave-one-protein-out jackknifing procedure, they were capable of reaching the top L/5 prediction accuracies of 49.5% and 48.8% for two different residue contact definitions, respectively. The predicted residue contacts were further employed to predict interacting helical pairs and achieved the Matthew's correlation coefficients of 0.430 and 0.424, according to two different residue contact definitions, respectively. To facilitate the academic community, the TMhhcp server has been made freely accessible at http://protein.cau.edu.cn/tmhhcp.

BACKGROUND: Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data. PRINCIPAL FINDINGS: Water proton T(1), T(2), and T(1rho) of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T(2) and T(1rho), but did not change high-field T(1). T(1rho) dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T(1rho)(0) values that closely approximated T(2). The T(1rho) dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T(1rho) dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz. CONCLUSIONS: Proton exchange is a minor pathway for tissue T(1) and T(1rho) relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T(1rho) to T(1).

Background: protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity. Methodology/Principal Findings: here, we use the AGGRESCAN algorithm to approximate the aggregation profile of an experimental cytosolic Escherichia coli proteome. The analysis indicates that the aggregation propensity of bacterial proteins is associated with their length, conformation, location, function, and abundance. The data are consistent with the predictions of other algorithms on different theoretical proteomes. Conclusions/Significance: overall, the study suggests that the avoidance of protein aggregation in functional environments acts as a strong evolutionary constraint on polypeptide sequences in both prokaryotic and eukaryotic organisms.

A robust marker to describe mass, hydrophobicity and polarizability distribution holds the key to deciphering structural and folding constraints within proteins. Since each of these distributions is inhomogeneous in nature, the construct should be sensitive in describing the patterns therein. We show, for the first time, that the hydrophobicity and polarizability distributions in protein interior follow fractal scaling. It is found that (barring 'all-alpha') all the major structural classes of proteins have an amount of unused hydrophobicity left in them. This amount of untapped hydrophobicity is observed to be greater in thermophilic proteins, than that in their (structurally aligned) mesophilic counterparts. 'All-beta'(thermophilic, mesophilic alike) proteins are found to have maximum amount of unused hydrophobicity, while 'all-alpha' proteins have been found to have minimum polarizability. A non-trivial dependency is observed between dielectric constant and hydrophobicity distributions within (alpha+beta) and 'all-alpha' proteins, whereas absolutely no dependency is found between them in the 'all-beta' class. This study proves that proteins are not as optimally packed as they are supposed to be. It is also proved that origin of alpha-helices are possibly not hydrophobic but electrostatic; whereas beta-sheets are predominantly hydrophobic in nature. Significance of this study lies in protein engineering studies; because it quantifies the extent of packing that ensures protein functionality. It shows that myths regarding protein interior organization might obfuscate our knowledge of actual reality. However, if the later is studied with a robust marker of strong mathematical basis, unknown correlations can still be unearthed; which help us to understand the nature of hydrophobicity, causality behind protein folding, and the importance of anisotropic electrostatics in stabilizing a highly complex structure named 'proteins'.

Most biological processes are regulated through complex networks of transient protein interactions where a globular domain in one protein recognizes a linear peptide from another, creating a relatively small contact interface. Although sufficient to ensure binding, these linear motifs alone are usually too short to achieve the high specificity observed, and additional contacts are often encoded in the residues surrounding the motif (i.e. the context). Here, we systematically identified all instances of peptide-mediated protein interactions of known three-dimensional structure and used them to investigate the individual contribution of motif and context to the global binding energy. We found that, on average, the context is responsible for roughly 20% of the binding and plays a crucial role in determining interaction specificity, by either improving the affinity with the native partner or impeding non-native interactions. We also studied and quantified the topological and energetic variability of interaction interfaces, finding a much higher heterogeneity in the context residues than in the consensus binding motifs. Our analysis partially reveals the molecular mechanisms responsible for the dynamic nature of peptide-mediated interactions, and suggests a global evolutionary mechanism to maximise the binding specificity. Finally, we investigated the viability of non-native interactions and highlight cases of potential cross-reaction that might compensate for individual protein failure and establish backup circuits to increase the robustness of cell networks.

IgGs from patients with multiple sclerosis and systemic lupus erythematosus (SLE) purified on MBP-Sepharose in contrast to canonical proteases hydrolyze effectively only myelin basic protein (MBP), but not many other tested proteins. Here we have shown for the first time that anti-MBP SLE IgGs hydrolyze nonspecific tri- and tetrapeptides with an extreme low efficiency and cannot effectively hydrolyze longer 20-mer nonspecific oligopeptides corresponding to antigenic determinants (AGDs) of HIV-1 integrase. At the same time, anti-MBP SLE IgGs efficiently hydrolyze oligopeptides corresponding to AGDs of MBP. All sites of IgG-mediated proteolysis of 21-and 25-mer encephalytogenic oligopeptides corresponding to two known AGDs of MBP were found by a combination of reverse-phase chromatography, TLC, and MALDI spectrometry. Several clustered major, moderate, and minor sites of cleavage were revealed in the case of 21- and 25-mer oligopeptides. The active sites of anti-MBP abzymes are localised on their light chains, while heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high affinity to MBP and specificity of this protein hydrolysis. The affinity of anti-MBP abzymes for intact MBP is approximately 1000-fold higher than for the oligopeptides. The data suggest that all oligopeptides interact mainly with the light chains of different monoclonal abzymes of total pool of IgGs, which possesses a lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific than globular protein and can occur in several sites.

Both sodium chloride and sodium sulfate are able to stabilize yeast frataxin, causing an overall increase of its thermodynamic stability curve, with a decrease in the cold denaturation temperature and an increase in the hot denaturation one. The influence of low concentrations of these two salts on yeast frataxin stability can be assessed by the application of a theoretical model based on scaled particle theory. First developed to figure out the mechanism underlying cold denaturation in water, this model is able to predict the stabilization of globular proteins provided by these two salts. The densities of the salt solutions and their temperature dependence play a fundamental role.

We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔGu H 2 0) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradation.

For proteins, the mechanical properties of the folded state are directly related to function, which generally entails conformational motion. Through sub-Angstrom resolution measurements of the AC mechanical susceptibility of a globular protein we describe a new fundamental materials property of the folded state. For increasing amplitude of the forcing, there is a reversible transition from elastic to viscoelastic response. At fixed frequency, the amplitude of the deformation is piecewise linear in the force, with different slopes in the elastic and viscoelastic regimes. Effectively, the protein softens beyond a yield point defined by this transition. We propose that ligand induced conformational changes generally operate in this viscoelastic regime, and that this is a universal property of the folded state.

At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin.

Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.

Here we present a systematic analysis of accessible surface areas and hydrogen bonds of 2554 globular proteins from four structural classes (all-α, all-β, α/β and α+β proteins) that is aimed to learn in which structural class the accessible surface area increases with increasing protein molecular mass more rapidly than in other classes, and what structural peculiarities are responsible for this effect. The beta structural class of proteins was found to be the leader, with the following possible explanations of this fact. First, in beta structural proteins, the fraction of residues not included in the regular secondary structure is the largest, and second, the accessible surface area of packaged elements of the beta-structure increases more rapidly with increasing molecular mass in comparison with the alpha-structure. Moreover, in the beta structure, the probability of formation of backbone hydrogen bonds is higher than that in the alpha helix for all residues of α+β proteins (the average probability is 0.73±0.01 for the beta-structure and 0.60±0.01 for the alpha-structure without proline) and α/β proteins, except for asparagine, aspartic acid, glycine, threonine, and serine (0.70±0.01 for the beta-structure and 0.60±0.01 for the alpha-structure without the proline residue). There is a linear relationship between the number of hydrogen bonds and the number of amino acid residues in the protein (Number of hydrogen bonds=0.678·number of residues-3.350).

BackgroundAmyloid fibril formation is the hallmark of many human diseases, including Alzheimer's disease, type II diabetes and amyloidosis. Amyloid fibrils deposit in the extracellular space and generally co-localize with the glycosaminoglycans (GAGs) of the basement membrane. GAGs have been shown to accelerate the formation of amyloid fibrils in vitro for a number of protein systems. The high number of data accumulated so far has created the grounds for the construction of a database on the effects of a number of GAGs on different proteins.Methodology/principal findingsIn this study, we have constructed such a database and have used a computational approach that uses a combination of single parameter and multivariate analyses to identify the main chemical factors that determine the GAG-induced acceleration of amyloid formation. We show that the GAG accelerating effect is mainly governed by three parameters that account for three-fourths of the observed experimental variability: the GAG sulfation state, the solute molarity, and the ratio of protein and GAG molar concentrations. We then combined these three parameters into a single equation that predicts, with reasonable accuracy, the acceleration provided by a given GAG in a given condition.Conclusions/significanceIn addition to shedding light on the chemical determinants of the protein:GAG interaction and to providing a novel mathematical predictive tool, our findings highlight the possibility that GAGs may not have such an accelerating effect on protein aggregation under the conditions existing in the basement membrane, given the values of salt molarity and protein:GAG molar ratio existing under such conditions.

BackgroundIn the last decade, the inhibition of protein-protein interactions (PPIs) has emerged from both academic and private research as a new way to modulate the activity of proteins. Inhibitors of these original interactions are certainly the next generation of highly innovative drugs that will reach the market in the next decade. However, in silico design of such compounds still remains challenging.Methodology/principal findingsHere we describe this particular PPI chemical space through the presentation of 2P2I(DB), a hand-curated database dedicated to the structure of PPIs with known inhibitors. We have analyzed protein/protein and protein/inhibitor interfaces in terms of geometrical parameters, atom and residue properties, buried accessible surface area and other biophysical parameters. The interfaces found in 2P2I(DB) were then compared to those of representative datasets of heterodimeric complexes. We propose a new classification of PPIs with known inhibitors into two classes depending on the number of segments present at the interface and corresponding to either a single secondary structure element or to a more globular interacting domain. 2P2I(DB) complexes share global shape properties with standard transient heterodimer complexes, but their accessible surface areas are significantly smaller. No major conformational changes are seen between the different states of the proteins. The interfaces are more hydrophobic than general PPI's interfaces, with less charged residues and more non-polar atoms. Finally, fifty percent of the complexes in the 2P2I(DB) dataset possess more hydrogen bonds than typical protein-protein complexes. Potential areas of study for the future are proposed, which include a new classification system consisting of specific families and the identification of PPI targets with high druggability potential based on key descriptors of the interaction.Conclusions2P2I database stores structural information about PPIs with known inhibitors and provides a useful tool for biologists to assess the potential druggability of their interfaces. The database can be accessed at http://2p2idb.cnrs-mrs.fr.

Molecular dynamics simulations of membrane proteins have provided deeper insights into their functions and interactions with surrounding environments at the atomic level. However, compared to solvation of globular proteins, building a realistic protein/membrane complex is still challenging and requires considerable experience with simulation software. Membrane Builder in the CHARMM-GUI website (http://www.charmm-gui.org) helps users to build such a complex system using a web browser with a graphical user interface. Through a generalized and automated building process including system size determination as well as generation of lipid bilayer, pore water, bulk water, and ions, a realistic membrane system with virtually any kinds and shapes of membrane proteins can be generated in 5 minutes to 2 hours depending on the system size. Default values that were elaborated and tested extensively are given in each step to provide reasonable options and starting points for both non-expert and expert users. The efficacy of Membrane Builder is illustrated by its applications to 12 transmembrane and 3 interfacial membrane proteins, whose fully equilibrated systems with three different types of lipid molecules (DMPC, DPPC, and POPC) and two types of system shapes (rectangular and hexagonal) are freely available on the CHARMM-GUI website. One of the most significant advantages of using the web environment is that, if a problem is found, users can go back and re-generate the whole system again before quitting the browser. Therefore, Membrane Builder provides the intuitive and easy way to build and simulate the biologically important membrane system.

Rapid, accurate prediction of protein structure from amino acid sequence would accelerate fields as diverse as drug discovery, synthetic biology and disease diagnosis. Massively improved prediction of protein structures has been driven by improving the prediction of the amino acid residues that contact in their 3D structure. For an average globular protein, around 92% of all residue pairs are non-contacting, therefore accurate prediction of only a small percentage of inter-amino acid distances could increase the number of constraints to guide structure determination. We have trained deep neural networks to predict inter-residue contacts and distances. Distances are predicted with an accuracy better than most contact prediction techniques. Addition of distance constraints improved de novo structure predictions for test sets of 158 protein structures, as compared to using the best contact prediction methods alone. Importantly, usage of distance predictions allows the selection of better models from the structure pool without a need for an external model assessment tool. The results also indicate how the accuracy of distance prediction methods might be improved further.