The effect of polymer length in liquid-liquid phase separation.

Understanding the thermodynamics that drive liquid-liquid phase separation (LLPS) is quite important given the number of diverse biomolecular systems undergoing this phenomenon. Many studies have focused on condensates of long polymers, but very few systems of short-polymer condensates have been observed and studied. Here, we study a short-polymer system of various lengths of poly-adenine RNA and peptides formed by the RGRGG sequence repeats to understand the underlying thermodynamics of LLPS. Using the recently developed COCOMO coarse-grained (CG) model, we predicted condensates for lengths as short as 5-10 residues, which was then confirmed by experiment, making this one of the smallest LLPS systems yet observed. A free-energy model reveals that the length dependence of condensation is driven primarily by entropy of confinement. The simplicity of this system will provide the basis for understanding more biologically realistic systems.

Direct generation of protein conformational ensembles via machine learning.

Dynamics and conformational sampling are essential for linking protein structure to biological function. While challenging to probe experimentally, computer simulations are widely used to describe protein dynamics, but at significant computational costs that continue to limit the systems that can be studied. Here, we demonstrate that machine learning can be trained with simulation data to directly generate physically realistic conformational ensembles of proteins without the need for any sampling and at negligible computational cost. As a proof-of-principle we train a generative adversarial network based on a transformer architecture with self-attention on coarse-grained simulations of intrinsically disordered peptides. The resulting model, idpGAN, can predict sequence-dependent coarse-grained ensembles for sequences that are not present in the training set demonstrating that transferability can be achieved beyond the limited training data. We also retrain idpGAN on atomistic simulation data to show that the approach can be extended in principle to higher-resolution conformational ensemble generation.

Unfolding and Aggregation Pathways of Variable Domains from Immunoglobulin Light Chains.

Light chain amyloidosis is the most common form of systemic amyloidosis. This disease is caused by the formation and deposition of amyloid fibers made from immunoglobulin light chains. Environmental conditions such as pH and temperature can affect protein structure and induce the development of these fibers. Several studies have shed light on the native state, stability, dynamics, and final amyloid state of these proteins; however, the initiation process and the fibril formation pathway remain poorly understood structurally and kinetically. To study this, we analyzed the unfolding and aggregation process of the 6aJL2 protein under acidic conditions, with temperature changes, and upon mutation, using biophysical and computational techniques. Our results suggest that the differences in amyloidogenicity displayed by 6aJL2 under these conditions are caused by traversing different aggregation pathways, including unfolded intermediates and the formation of oligomers.

Modeling Concentration-dependent Phase Separation Processes Involving Peptides and RNA via Residue-Based Coarse-Graining.

Biomolecular condensation, especially liquid-liquid phase separation, is an important physical process with relevance for a number of different aspects of biological functions. Key questions of what drives such condensation, especially in terms of molecular composition, can be addressed via computer simulations, but the development of computationally efficient yet physically realistic models has been challenging. Here, the coarse-grained model COCOMO is introduced that balances the polymer behavior of peptides and RNA chains with their propensity to phase separate as a function of composition and concentration. COCOMO is a residue-based model that combines bonded terms with short- and long-range terms, including a Debye-Hückel solvation term. The model is highly predictive of experimental data on phase-separating model systems. It is also computationally efficient and can reach the spatial and temporal scales on which biomolecular condensation is observed with moderate computational resources.

Characterizing Transient Protein-Protein Interactions by Trp-Cys Quenching and Computer Simulations.

Transient protein-protein interactions occur frequently under the crowded conditions encountered in biological environments. Tryptophan-cysteine quenching is introduced as an experimental approach with minimal labeling for characterizing such interactions between proteins due to its sensitivity to nano- to microsecond dynamics on subnanometer length scales. The experiments are paired with computational modeling at different resolutions including fully atomistic molecular dynamics simulations for interpretation of the experimental observables and to gain molecular-level insights. This approach is applied to model systems, villin variants and the drkN SH3 domain, in the presence of protein G crowders. It is demonstrated that Trp-Cys quenching experiments can differentiate between overall attractive and repulsive interactions between different proteins, and they can discern variations in interaction preferences at different protein surface locations. The close integration between experiment and simulations also provides an opportunity to evaluate different molecular force fields for the simulation of concentrated protein solutions.

Site-Specific Interactions with Copper Promote Amyloid Fibril Formation for λ6aJL2-R24G.

Light-chain amyloidosis (AL) is one of the most common systemic amyloidoses, and it is characterized by the deposition of immunoglobulin light chain (LC) variable domains as insoluble amyloid fibers in vital organs and tissues. The recombinant protein 6aJL2-R24G contains λ6a and JL2 germline genes and also contains the Arg24 by Gly substitution. This mutation is present in 25% of all amyloid-associated λ6 LC cases, reduces protein stability, and increases the propensity to form amyloid fibers. In this study, it was found that the interaction of 6aJL2-R24G with Cu(II) decreases the thermal stability of the protein and accelerates the amyloid fibril formation, as observed by fluorescence spectroscopy. Isothermal calorimetry titration showed that Cu(II) binds to the protein with micromolar affinity. His99 may be one of the main Cu(II) interaction sites, as observed by nuclear magnetic resonance spectroscopy. The binding of Cu(II) to His99 induces larger fluctuations of the CDR1 and loop C″, as shown by molecular dynamics simulations. Thus, Cu(II) binding may be inducing the loss of interactions between CDR3 and CDR1, making the protein less stable and more prone to form amyloid fibers. This study provides insights into the mechanism of metal-induced aggregation of the 6aJL2-R24G protein and sheds light on the bio-inorganic understanding of AL disease.

Different Dynamics in 6aJL2 Proteins Associated with AL Amyloidosis, a Conformational Disease.

Light-chain amyloidosis (AL) is the most common systemic amyloidosis and is caused by the deposition of mainly insoluble immunoglobulin light chain amyloid fibrils in multiple organs, causing organ failure and eventually death. The germ-line λ6a has been implicated in AL, where a single point mutant at amino acid 24 (6aJL2-R24G) has been observed in around 25% of patient samples. Structural analysis has shown only subtle differences between both proteins; nevertheless, 6aJL2-R24G is more prone to form amyloid fibrils. To improve our understanding of the role of protein flexibility in amyloid fibril formation, we have used a combination of solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to complement the structural insight with dynamic knowledge. Fast timescale dynamics (ps-ns) were equivalent for both proteins, but suggested exchange events for some residues. Even though most of the intermediate dynamics (µs-ms) occurred at a similar region for both proteins, the specific characteristics are very different. A minor population detected in the dispersion experiments could be associated with the formation of an off-pathway intermediate that protects from fiber formation more efficiently in the germ-line protein. Moreover, we found that the hydrogen bond patterns for both proteins are similar, but the lifetime for the mutant is significantly reduced; as a consequence, there is a decrease in the stability of the tertiary structure that extends throughout the protein and leads to an increase in the propensity to form amyloid fibers.

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0 pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0 pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors.

The Hcp-like protein HilE inhibits homodimerization and DNA binding of the virulence-associated transcriptional regulator HilD in Salmonella.

HilD is an AraC-like transcriptional regulator that plays a central role in Salmonella virulence. HilD controls the expression of the genes within the Salmonella pathogenicity island 1 (SPI-1) and of several genes located outside SPI-1, which are mainly required for Salmonella invasion of host cells. The expression, amount, and activity of HilD are tightly controlled by the activities of several factors. The HilE protein represses the expression of the SPI-1 genes through its interaction with HilD; however, the mechanism by which HilE affects HilD is unknown. In this study, we used genetic and biochemical assays revealing how HilE controls the transcriptional activity of HilD. We found that HilD needs to assemble in homodimers to induce expression of its target genes. Our results further indicated that HilE individually interacts with each the central and the C-terminal HilD regions, mediating dimerization and DNA binding, respectively. We also observed that these interactions consistently inhibit HilD dimerization and DNA binding. Interestingly, a computational analysis revealed that HilE shares sequence and structural similarities with Hcp proteins, which act as structural components of type 6 secretion systems in Gram-negative bacteria. In conclusion, our results uncover the molecular mechanism by which the Hcp-like protein HilE controls dimerization and DNA binding of the virulence-promoting transcriptional regulator HilD. Our findings may indicate that HilE's activity represents a functional adaptation during the evolution of Salmonella pathogenicity.

Convergent mechanisms favor fast amyloid formation in two lambda 6a Ig light chain mutants.

Extracellular deposition as amyloids of immunoglobulin light chains causes light chain amyloidosis. Among the light chain families, lambda 6a is one of the most frequent in light chain amyloidosis patients. Its germline protein, 6aJL2, and point mutants, R24G and P7S, are good models to study fibrillogenesis, because their stability and fibril formation characteristics have been described. Both mutations make the germline protein unstable and speed up its ability to aggregate. To date, there is no molecular mechanism that explains how these differences in amyloidogenesis can arise from a single mutation. To look into the structural and dynamical differences in the native state of these proteins, we carried out molecular dynamics simulations at room temperature. Despite the structural similarity of the germline protein and the mutants, we found differences in their dynamical signatures that explain the mutants' increased tendency to form amyloids. The contact network alterations caused by the mutations, though different, converge in affecting two anti-aggregation motifs present in light chain variable domains, suggesting a different starting point for aggregation in lambda chains compared to kappa chains.

A family 13 thioesterase isolated from an activated sludge metagenome: Insights into aromatic compounds metabolism.

Activated sludge is produced during the treatment of sewage and industrial wastewaters. Its diverse chemical composition allows growth of a large collection of microbial phylotypes with very different physiologic and metabolic profiles. Thus, activated sludge is considered as an excellent environment to discover novel enzymes through functional metagenomics, especially activities related with degradation of environmental pollutants. Metagenomic DNA was isolated and purified from an activated sludge sample. Metagenomic libraries were subsequently constructed in Escherichia coli. Using tributyrin hydrolysis, a screening by functional analysis was conducted and a clone that showed esterase activity was isolated. Blastx analysis of the sequence of the cloned DNA revealed, among others, an ORF that encodes a putative thioesterase with 47-64% identity to GenBank CDS reported genes, similar to those in the hotdog fold thioesterase superfamily. On the basis of its amino acid similarity and its homology-modelled structure we deduced that this gene encodes an enzyme (ThYest_ar) that belongs to family TE13, with a preference for aryl-CoA substrates and a novel catalytic residue constellation. Plasmid retransformation in E. coli confirmed the clone's phenotype, and functional complementation of a paaI E. coli mutant showed preference for phenylacetate over chlorobenzene as a carbon source. This work suggests a role for TE13 family thioesterases in swimming and degradation approaches for phenyl acetic acid. Proteins 2017; 85:1222-1237. © 2017 Wiley Periodicals, Inc.

Inhibition of Light Chain 6aJL2-R24G Amyloid Fiber Formation Associated with Light Chain Amyloidosis.

Light chain amyloidosis (AL) is a deadly disease characterized by the deposition of monoclonal immunoglobulin light chains as insoluble amyloid fibrils in different organs and tissues. Germ line λ VI has been closely related to this condition; moreover, the R24G mutation is present in 25% of the proteins of this germ line in AL patients. In this work, five small molecules were tested as inhibitors of the formation of amyloid fibrils from the 6aJL2-R24G protein. We have found by thioflavin T fluorescence and transmission electron microscopy that EGCG inhibits 6aJL2-R24G fibrillogenesis. Furthermore, using nuclear magnetic resonance spectroscopy, dynamic light scattering, and isothermal titration calorimetry, we have determined that the inhibition is due to binding to the protein in its native state, interacting mainly with aromatic residues.

Identification of a novel carbohydrate esterase from Bjerkandera adusta: structural and function predictions through bioinformatics analysis and molecular modeling.

A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg(-1) protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin.