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TAR DNA-binding protein 43 (TDP-43) is a multidomain protein involved in the regulation of RNA metabolism, and its aggregates have been observed in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Numerous studies indicate TDP-43 can undergo liquid-liquid phase separation (LLPS) in vitro and is a component of biological condensates. Homo-oligomerization via the folded N-terminal domain (aa:1-77) and the conserved helical region (aa:319-341) of the disordered, C-terminal domain is found to be an important driver of TDP-43 phase separation. However, a comprehensive molecular view of TDP-43 phase separation, particularly regarding the nature of heterodomain interactions, is lacking due to the challenges associated with its stability and purification. Here, we utilize all-atom and coarse-grained (CG) molecular dynamics (MD) simulations to uncover the network of interdomain interactions implicated in TDP-43 phase separation. All-atom simulations uncovered the presence of transient, interdomain interactions involving flexible linkers, RNA-recognition motif (RRM) domains and a charged segment of disordered C-terminal domain (CTD). CG simulations indicate these inter-domain interactions which affect the conformational landscape of TDP-43 in the dilute phase are also prevalent in the condensed phase. Finally, sequence and surface charge distribution analysis coupled with all-atom simulations (at high salt) confirmed that the transient interdomain contacts are predominantly electrostatic in nature. Overall, our findings from multiscale simulations lead to a greater appreciation of the complex interaction network underlying the structural landscape and phase separation of TDP-43., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Material properties of phase-separated biomolecular assemblies, enriched with disordered proteins, dictate their ability to participate in many cellular functions. Despite the significant effort dedicated to understanding how the sequence of the disordered protein drives its phase separation to form condensates, little is known about the sequence determinants of condensate material properties. Here, we computationally decipher these relationships for charged disordered proteins using model sequences comprised of glutamic acid and lysine residues as well as naturally occurring sequences of LAF1's RGG domain and DDX4's N-terminal domain. We do so by delineating how the arrangement of oppositely charged residues within these sequences influences the dynamical, rheological, and interfacial properties of the condensed phase through equilibrium and non-equilibrium molecular simulations using the hydropathy scale and Martini models. Our computations yield material properties that are quantitatively comparable with experimentally characterized condensate systems. Interestingly, we find that the material properties of both the model and natural proteins respond similarly to the segregation of charges, despite their very different sequence compositions. Condensates of the highly charge-segregated sequences exhibit slower dynamics than the uniformly charge-patterned sequences, because of their comparatively long-lived molecular contacts between oppositely charged residues. Surprisingly, the molecular interactions within the condensate are highly similar to those within a single-chain for all sequences. Consequently, the condensate material properties of charged disordered proteins are strongly correlated with their dense phase contact dynamics and their single-chain structural properties. Our findings demonstrate the potential to harness the sequence characteristics of disordered proteins for predicting and engineering the material properties of functional condensates, with insights from the dilute phase properties.

The heterochromatin protein 1 (HP1) family is a crucial component of heterochromatin with diverse functions in gene regulation, cell cycle control, and cell differentiation. In humans, there are three paralogs, HP1α, HP1β, and HP1γ, which exhibit remarkable similarities in their domain architecture and sequence properties. Nevertheless, these paralogs display distinct behaviors in liquid-liquid phase separation (LLPS), a process linked to heterochromatin formation. Here, we employ a coarse-grained simulation framework to uncover the sequence features responsible for the observed differences in LLPS. We highlight the significance of the net charge and charge patterning along the sequence in governing paralog LLPS propensities. We also show that both highly conserved folded and less-conserved disordered domains contribute to the observed differences. Furthermore, we explore the potential co-localization of different HP1 paralogs in multicomponent assemblies and the impact of DNA on this process. Importantly, our study reveals that DNA can significantly reshape the stability of a minimal condensate formed by HP1 paralogs due to competitive interactions of HP1α with HP1β and HP1γ versus DNA. In conclusion, our work highlights the physicochemical nature of interactions that govern the distinct phase-separation behaviors of HP1 paralogs and provides a molecular framework for understanding their role in chromatin organization., Competing Interests: Competing Interests: All authors declare they have no competing interests.

The Polycomb Group (PcG) complex PRC1 represses transcription, forms condensates in cells, and modifies chromatin architecture. These processes are connected through the essential, polymerizing Sterile Alpha Motif (SAM) present in the PRC1 subunit Polyhomeotic (Ph). In vitro , Ph SAM drives formation of short oligomers and phase separation with DNA or chromatin in the context of a Ph truncation ("mini-Ph"). Oligomer length is controlled by the long disordered linker (L) that connects the SAM to the rest of Ph--replacing Drosophila PhL with the evolutionarily diverged human PHC3L strongly increases oligomerization. How the linker controls SAM polymerization, and how polymerization and the linker affect condensate formation are not know. We analyzed PhL and PHC3L using biochemical assays and molecular dynamics (MD) simulations. PHC3L promotes mini-Ph phase separation and makes it relatively independent of DNA. In MD simulations, basic amino acids in PHC3L form contacts with acidic amino acids in the SAM. Engineering the SAM to make analogous charge-based contacts with PhL increased polymerization and phase separation, partially recapitulating the effects of the PHC3L. Ph to PHC3 linker swaps and SAM surface mutations alter Ph condensate formation in cells, and Ph function in Drosophila imaginal discs. Thus, SAM-driven phase separation and polymerization are conserved between flies and mammals, but the underlying mechanisms have diverged through changes to the disordered linker., Competing Interests: Declaration of interests The authors declare no competing interests.

Recent advances in coarse-grained (CG) computational models for DNA have enabled molecular-level insights into the behavior of DNA in complex multiscale systems. However, most existing CG DNA models are not compatible with CG protein models, limiting their applications for emerging topics such as protein-nucleic acid assemblies. Here, we present a new computationally efficient CG DNA model. We first use experimental data to establish the model's ability to predict various aspects of DNA behavior, including melting thermodynamics and relevant local structural properties such as the major and minor grooves. We then employ an all-atom hydropathy scale to define non-bonded interactions between protein and DNA sites, to make our DNA model compatible with an existing CG protein model (HPS-Urry), that is extensively used to study protein phase separation, and show that our new model reasonably reproduces the experimental binding affinity for a prototypical protein-DNA system. To further demonstrate the capabilities of this new model, we simulate a full nucleosome with and without histone tails, on a microsecond timescale, generating conformational ensembles and provide molecular insights into the role of histone tails in influencing the liquid-liquid phase separation (LLPS) of HP1α proteins. We find that histone tails interact favorably with DNA, influencing the conformational ensemble of the DNA and antagonizing the contacts between HP1α and DNA, thus affecting the ability of DNA to promote LLPS of HP1α. These findings shed light on the complex molecular framework that fine-tunes the phase transition properties of heterochromatin proteins and contributes to heterochromatin regulation and function. Overall, the CG DNA model presented here is suitable to facilitate micron-scale studies with sub-nm resolution in many biological and engineering applications and can be used to investigate protein-DNA complexes, such as nucleosomes, or LLPS of proteins with DNA, enabling a mechanistic understanding of how molecular information may be propagated at the genome level., Competing Interests: Conflicts of Interest The authors declare no competing financial interest.

Interaction strength and localization are critical parameters controlling the single-chain and condensed-state properties of intrinsically disordered proteins (IDPs). Here, we decipher these relationships using coarse-grained heteropolymers comprised of hydrophobic (H) and polar (P) monomers as model IDPs. We systematically vary the fraction of P monomers X P and employ two distinct particle-based models that include either strong localized attractions between only H-H pairs (HP model) or weak distributed attractions between both H-H and H-P pairs (HP+ model). To compare different sequences and models, we first carefully tune the attraction strength for all sequences to match the single-chain radius of gyration. Interestingly, we find that this procedure produces similar conformational ensembles, nonbonded potential energies, and chain-level dynamics for single chains of almost all sequences in both models, with some deviations for the HP model at large X P . However, we observe a surprisingly rich phase behavior for the sequences in both models that deviates from the expectation that similarity at the single-chain level will translate to a similar phase-separation propensity. Coexistence between dilute and dense phases is only observed up to a model-dependent X P , despite the presence of favorable interchain interactions, which we quantify using the second virial coefficient. Instead, the limited number of attractive sites (H monomers) leads to the self-assembly of finite-sized clusters of different sizes depending on X P . Our findings strongly suggest that models with distributed interactions favor the formation of liquid-like condensates over a much larger range of sequence compositions compared to models with localized interactions.

Nature uses chromophore networks, with highly optimized structural and energetic characteristics, to perform important chemical functions. Due to its modularity, predictable aggregation characteristics, and established synthetic protocols, structural DNA nanotechnology is a promising medium for arranging chromophore networks with analogous structural and energetic controls. However, this high level of control creates a greater need to know how to optimize the systems precisely. This study uses the system's modularity to produce variations of a coupled 14-Site chromophore network. It uses machine-learning algorithms and spectroscopy measurements to reveal the energy-transport roles of these Sites, paying particular attention to the cooperative and inhibitive effects they impose on each other for transport across the network. The physical significance of these patterns is contextualized, using molecular dynamics simulations and energy-transport modeling. This analysis yields insights about how energy transfers across the Donor-Relay and Relay-Acceptor interfaces, as well as the energy-transport pathways through the homogeneous Relay segment. Overall, this report establishes an approach that uses machine-learning methods to understand, in fine detail, the role that each Site plays in an optoelectronic molecular network.

Heterochromatin protein 1α (HP1α) is a crucial element of chromatin organization. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can undergo phase separation upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. Here, we combine computational and experimental approaches to elucidate the molecular interactions that drive these processes. In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged HP1α peptide ligands and disrupted by the addition of negatively charged or neutral peptides. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in HP1α LLPS where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine-tune the properties of HP1α and that can contribute to heterochromatin regulation and function., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The stability and physiological function of many biomolecular coacervates depend on the structure and dynamics of intrinsically disordered proteins (IDPs) that typically contain a significant fraction of charged residues. Although the effect of relative arrangement of charged residues on IDP conformation is a well-studied problem, the associated changes in dynamics are far less understood. In this work, we systematically interrogate the effects of charge distribution on the chain-level and segmental dynamics of polyampholytic IDPs in dilute solutions. We study a coarse-grained model polyampholyte consisting of an equal fraction of two oppositely charged residues (glutamic acid and lysine) that undergoes a transition from an ideal chain-like conformation for uniformly charge-patterned sequences to a semi-compact conformation for highly charge-segregated sequences. Changes in the chain-level dynamics with increasing charge segregation correlate with changes in conformation. The chain-level and segmental dynamics conform to simple homopolymer models for uniformly charge-patterned sequences but deviate with increasing charge segregation, both in the presence and absence of hydrodynamic interactions. We discuss the significance of these findings, obtained for a model polyampholyte, in the context of a charge-rich intrinsically disordered region of the naturally occurring protein LAF-1. Our findings have important implications for understanding the effects of charge patterning on the dynamics of polyampholytic IDPs in dilute conditions using polymer scaling theories.

Dye aggregates and their excitonic properties are of interest for their applications to organic photovoltaics, non-linear optics, and quantum information systems. DNA scaffolding has been shown to be effective at promoting the aggregation of dyes in a controllable manner. Specifically, isolated DNA Holliday junctions have been used to achieve strongly coupled cyanine dye dimers. However, the structural properties of the dimers and the DNA, as well as the role of Holliday junction isomerization are not fully understood. To study the dynamics of cyanine dimers in DNA, molecular dynamics simulations were carried out for adjacent and transverse dimers attached to Holliday junctions in two different isomers. It was found that dyes attached to adjacent strands in the junction exhibit stronger dye-DNA interactions and larger inter-dye separations compared to transversely attached dimers, as well as end-to-end arrangements. Transverse dimers exhibit lower inter-dye separations and more stacked configurations. Furthermore, differences in Holliday junction isomer are analyzed and compared to dye orientations. For transverse dyes exhibiting the smaller inter-dye separations, excitonic couplings were calculated and shown to be in agreement with experiment. Our results suggested that dye attachment locations on DNA Holliday junctions affect dye-DNA interactions, dye dynamics, and resultant dye orientations which can guide the design of DNA-templated cyanine dimers with desired properties., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Recent advances in residue-level coarse-grained (CG) computational models have enabled molecular-level insights into biological condensates of intrinsically disordered proteins (IDPs), shedding light on the sequence determinants of their phase separation. The existing CG models that treat protein chains as flexible molecules connected via harmonic bonds cannot populate common secondary-structure elements. Here, we present a CG dihedral angle potential between four neighboring beads centered at C α atoms to faithfully capture the transient helical structures of IDPs. In order to parameterize and validate our new model, we propose C α -based helix assignment rules based on dihedral angles that succeed in reproducing the atomistic helicity results of a polyalanine peptide and folded proteins. We then introduce sequence-dependent dihedral angle potential parameters (ε d ) and use experimentally available helical propensities of naturally occurring 20 amino acids to find their optimal values. The single-chain helical propensities from the CG simulations for commonly studied prion-like IDPs are in excellent agreement with the NMR-based α-helix fraction, demonstrating that the new HPS-SS model can accurately produce structural features of IDPs. Furthermore, this model can be easily implemented for large-scale assembly simulations due to its simplicity.

In the finely regulated process of mammalian erythropoiesis, the path of the labile iron pool into mitochondria for heme production is not well understood. Existing models for erythropoiesis do not include a central role for the ubiquitous iron storage protein ferritin; one model proposes that incoming endosomal Fe 3+ bound to transferrin enters the cytoplasm through an ion transporter after reduction to Fe 2+ and is taken up into mitochondria through mitoferrin-1 transporter. Here, we apply a dual three-dimensional imaging and spectroscopic technique, based on scanned electron probes, to measure Fe 3+ in ex vivo human hematopoietic stem cells. After seven days in culture, we observe cells displaying a highly specialized architecture with anchored clustering of mitochondria and massive accumulation of nanoparticles containing high iron concentrations localized to lysosomal storage depots, identified as ferritin. We hypothesize that lysosomal ferritin iron depots enable continued heme production after expulsion of most of the cellular machinery., Competing Interests: The authors declare no competing financial interests.

We present improvements to the hydropathy scale (HPS) coarse-grained (CG) model for simulating sequence-specific behavior of intrinsically disordered proteins (IDPs), including their liquid-liquid phase separation (LLPS). The previous model based on an atomistic hydropathy scale by Kapcha and Rossky (KR scale) is not able to capture some well-known LLPS trends such as reduced phase separation propensity upon mutations (R-to-K and Y-to-F). Here, we propose to use the Urry hydropathy scale instead, which was derived from the inverse temperature transitions in a model polypeptide with guest residues X. We introduce two free parameters to shift (Δ) and scale (µ) the overall interaction strengths for the new model (HPS-Urry) and use the experimental radius of gyration for a diverse group of IDPs to find their optimal values. Interestingly, many possible (Δ, µ) combinations can be used for typical IDPs, but the phase behavior of a low-complexity (LC) sequence FUS is only well described by one of these models, which highlights the need for a careful validation strategy based on multiple proteins. The CG HPS-Urry model should enable accurate simulations of protein LLPS and provide a microscopically detailed view of molecular interactions., (© 2021 The Protein Society.)

Biomolecules undergo liquid-liquid phase separation (LLPS), resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post-translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed-charge atomistic force fields, we parameterize the size and atomistic hydropathy of the coarse-grained-modified amino acid beads and, hence, the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the protein's single-chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of Fused in Sarcoma (FUS) and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution., (Copyright © 2021 Biophysical Society. All rights reserved.)

The formation of membraneless organelles in cells commonly occurs via liquid-liquid phase separation (LLPS) and is in many cases driven by multivalent interactions between intrinsically disordered proteins (IDPs). Investigating the nature of these interactions, and their effect on dynamics within the condensed phase, is therefore of critical importance but very challenging for either simulation or experiment. Here, we study these interactions and their dynamics by pairing a novel multiscale simulation strategy with microsecond all-atom MD simulations of a condensed, IDP-rich phase. We simulate two IDPs this way, the low complexity domain of FUS and the N-terminal disordered domain of LAF-1, and find good agreement with experimental information about average density, water content, and residue-residue contacts. We go significantly beyond what is known from experiments by showing that ion partitioning within the condensed phase is largely driven by the charge distribution of the proteins and-in the cases considered-shows little evidence of preferential interactions of the ions with the proteins. Furthermore, we can probe the microscopic diffusive dynamics within the condensed phase, showing that water and ions are in dynamic equilibrium between dense and dilute phases, and their diffusion is reduced in the dense phase. Despite their high concentration in the condensate, the protein molecules also remain mobile, explaining the observed liquid-like properties of this phase. We finally show that IDP self-association is driven by a combination of nonspecific hydrophobic interactions as well as hydrogen bonds, salt bridges, and π-π and cation-π interactions. The simulation approach presented here allows the structural and dynamical properties of biomolecular condensates to be studied in microscopic detail and is generally applicable to single- and multicomponent systems of proteins and nucleic acids involved in LLPS.

Ribonucleoprotein (RNP) granules are membraneless organelles (MLOs), which majorly consist of RNA and RNA-binding proteins and are formed via liquid-liquid phase separation (LLPS). Experimental studies investigating the drivers of LLPS have shown that intrinsically disordered proteins (IDPs) and nucleic acids like RNA and other polynucleotides play a key role in modulating protein phase separation. There is currently a dearth of modelling techniques which allow one to delve deeper into how polynucleotides play the role of a modulator/promoter of LLPS in cells using computational methods. Here, we present a coarse-grained polynucleotide model developed to fill this gap, which together with our recently developed HPS model for protein LLPS, allows us to capture the factors driving protein-polynucleotide phase separation. We explore the capabilities of the modelling framework with the LAF-1 RGG system which has been well studied in experiments and also with the HPS model previously. Further taking advantage of the fact that the HPS model maintains sequence specificity we explore the role of charge patterning on controlling polynucleotide incorporation into condensates. With increased charge patterning we observe formation of structured or patterned condensates which suggests the possible roles of polynucleotides in not only shifting the phase boundaries but also introducing microscopic organization in MLOs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Understanding the conformational ensemble of an intrinsically disordered protein (IDP) is of great interest due to its relevance to critical intracellular functions and diseases. It is now well established that the polymer scaling behavior can provide a great deal of information about the conformational properties as well as liquid-liquid phase separation of an IDP. It is, therefore, extremely desirable to be able to predict an IDP's scaling behavior from the protein sequence itself. The work in this direction so far has focused on highly charged proteins and how charge patterning can perturb their structural properties. As naturally occurring IDPs are composed of a significant fraction of uncharged amino acids, the rules based on charge content and patterning are only partially helpful in solving the problem. Here, we propose a new order parameter, sequence hydropathy decoration, which can provide a near-quantitative understanding of scaling and structural properties of IDPs devoid of charged residues. We combine this with a charge patterning parameter, sequence charge decoration, to obtain a general equation, parametrized from extensive coarse-grained simulation data, for predicting protein dimensions from the sequence. We finally test this equation against available experimental data and find a semiquantitative match in predicting the scaling behavior. We also provide guidance on how to extend this approach to experimental data, which should be feasible in the near future.

Proteins that undergo liquid-liquid phase separation (LLPS) have been shown to play a critical role in many physiological functions through formation of condensed liquid-like assemblies that function as membraneless organelles within biological systems. To understand how different proteins may contribute differently to these assemblies and their functions, it is important to understand the molecular driving forces of phase separation and characterize their phase boundaries and material properties. Experimental studies have shown that intrinsically disordered regions of these proteins are a major driving force, as many of them undergo LLPS in isolation. Previous work on polymer solution phase behavior suggests a potential correspondence between intramolecular and intermolecular interactions that can be leveraged to discover relationships between single-molecule properties and phase boundaries. Here, we take advantage of a recently developed coarse-grained framework to calculate the θ temperature [Formula: see text], the Boyle temperature [Formula: see text], and the critical temperature [Formula: see text] for 20 diverse protein sequences, and we show that these three properties are highly correlated. We also highlight that these correlations are not specific to our model or simulation methodology by comparing between different pairwise potentials and with data from other work. We, therefore, suggest that smaller simulations or experiments to determine [Formula: see text] or [Formula: see text] can provide useful insights into the corresponding phase behavior., Competing Interests: The authors declare no conflict of interest.

Unlike dilute experimental conditions under which biological molecules are typically characterized, the cell interior is crowded by macromolecules, which affects both the thermodynamics and kinetics of in vivo processes. Although the excluded-volume effects of macromolecular crowding are expected to cause compaction of unfolded and disordered proteins, the extent of this effect is uncertain. We use a coarse-grained model to represent proteins with varying sequence content and directly observe changes in chain dimensions in the presence of purely repulsive spherical crowders. We find that the extent of crowding-induced compaction is dependent not only on crowder size and concentration, but also on the properties of the protein itself. In fact, we observe a nonmonotonic trend between the dimensions of the polypeptide chain in bulk and the degree of compaction: the most extended chains experience up to 24% compaction, the most compact chains show virtually no change, and intermediate chains compress by up to 40% in size at a 40% crowder volume fraction. Free-volume theory combined with an impenetrable ellipsoidal representation of the chains predicts the crowding effects only for collapsed protein chains. An additional scaling factor, which can be easily computed from protein-crowder potential of mean force, corrects for the penetrability of extended chains and is sufficient to capture the observed nonmonotonic trend in compaction., (Copyright © 2016 Biophysical Society. All rights reserved.)

Protein-protein interactions are important in many essential biological functions, such as transcription, translation, and signal transduction. Much progress has been made in understanding protein-protein association in dilute solution via experimentation and simulation. Cells, however, contain various macromolecules, such as DNA, RNA, proteins, among many others, and a myriad of non-specific interactions (usually weak) are present between these cellular constituents. In this review article, we describe the important developments in recent years that have furthered our understanding and even allowed prediction of the consequences of macromolecular crowding on protein-protein interactions. We outline the development of our crowding theory that can predict the change in binding free energy due to crowding quantitatively for both repulsive and attractive protein-crowder interactions. One of the most important findings from our recent work is that weak attractive interactions between crowders and proteins can actually destabilize protein complex formation as opposed to the commonly assumed stabilizing effect predicted based on traditional crowding theories that only account for the entropic-excluded volume effects. We also discuss the implications of macromolecular crowding on the population of encounter versus specific native complex.

We studied the significance of alterations in the metabolomics of sulfur-containing substances in rapidly regenerating rat livers. Male rats were subjected to two-thirds partial hepatectomy (PHx), and the changes in hepatic levels of major sulfur-containing amino acids and related substances were monitored for 2 weeks. Liver weight began to increase from 24 h after the surgery, and appeared to recover fully in 2 weeks. Serum alanine aminotransferase and aspartate aminotransferase activities were elevated immediately after the surgery and returned slowly to normal levels in 2 weeks. Methionine, S-adenosylmethionine (SAM), cystathionine and cysteine were increased rapidly and remained elevated for longer than 1 week. Hepatic glutathione concentration was increased gradually for 24 h, and then decreased thereafter, whereas hypotaurine was elevated drastically right after the surgery. Hepatic concentrations of polyamines were altered significantly by PHx. In the hepatectomized livers putrescine concentration was elevated rapidly, reaching a level 40- to 50-fold greater than normal in 6-12 h. Ornithine, the metabolic substrate for putrescine synthesis, was also elevated markedly. Spermidine was increased significantly, whereas spermine was depressed below normal, which appeared to be due to the increased consumption of decarboxylated SAM for spermidine biosynthesis. The results show that the metabolomics of sulfur-containing amino acids and related substances is altered profoundly in regenerating rat livers until the original weight is recovered. Hepatic concentrations of polyamines after PHx are closely associated with the alteration in the metabolomics of sulfur-containing substances. The implication of these changes in the progression of liver regeneration is discussed.

Cytochrome c oxidase is an efficient energy transducer that reduces oxygen to water and converts the released chemical energy into an electrochemical membrane potential. As a true proton pump, cytochrome c oxidase translocates protons across the membrane against this potential. Based on a wealth of experiments and calculations, an increasingly detailed picture of the reaction intermediates in the redox cycle has emerged. However, the fundamental mechanism of proton pumping coupled to redox chemistry remains largely unresolved. Here we examine and extend a kinetic master-equation approach to gain insight into redox-coupled proton pumping in cytochrome c oxidase. Basic principles of the cytochrome c oxidase proton pump emerge from an analysis of the simplest kinetic models that retain essential elements of the experimentally determined structure, energetics, and kinetics, and that satisfy fundamental physical principles. The master-equation models allow us to address the question of how pumping can be achieved in a system in which all reaction steps are reversible. Whereas proton pumping does not require the direct modulation of microscopic reaction barriers, such kinetic gating greatly increases the pumping efficiency. Further efficiency gains can be achieved by partially decoupling the proton uptake pathway from the active-site region. Such a mechanism is consistent with the proposed Glu valve, in which the side chain of a key glutamic acid shuttles between the D channel and the active-site region. We also show that the models predict only small proton leaks even in the absence of turnover. The design principles identified here for cytochrome c oxidase provide a blueprint for novel biology-inspired fuel cells, and the master-equation formulation should prove useful also for other molecular machines. ., (Published by Elsevier B.V.)

We study the effects of attractive interactions between spherical crowders and protein residues on the thermodynamics and structure of two weakly binding protein complexes: ubiquitin/UIM1 and cytochrome c/cytochrome c peroxidase. Systematic replica exchange Monte Carlo (REMC) simulations are performed over a range of attraction strengths and crowder packing fractions using a transferable coarse-grained protein binding model. We find that moderate attractive interactions (≈0.2 kcal/mol) between crowders and protein residues can destabilize protein association, and therefore counteract the stabilizing effect of excluded volume interactions. The destabilization of protein binding, as measured by an increase in binding free energy, increases with increasing crowder packing fraction. For a critical attraction strength value, which is found to be approximately independent of crowder packing fraction, the destabilization due to attractions is exactly canceled by the stabilization effect of excluded volume interactions. This results in a net zero change in binding free energy with respect to a crowder-free solution. Further, we find that attractive interactions between crowders and protein residues can favor transiently bound encounter complexes over the native specific complexes in the bound state. We propose a simple theoretical model based on the scaled particle theory augmented by a mean-field attraction term that can explain our simulation results semiquantitatively., (© 2011 American Chemical Society)

Time-dependent external perturbations provide powerful probes of the function of molecular machines. Here we study biological proton pumping in an oscillating electric field. The protein cytochrome c oxidase is the main energy transducer in aerobic life, converting chemical energy into an electric potential by pumping protons across a membrane. With the help of master-equation descriptions that recover the key thermodynamic and kinetic properties of this biological "fuel cell," we show that the proton pumping efficiency and the electronic currents in steady state depend significantly on the frequency and amplitude of the applied field, allowing us to distinguish between different microscopic mechanisms of the machine. A spectral analysis reveals dominant reaction steps consistent with an electron-gated pumping mechanism.

Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, reduces oxygen to water and uses the released energy to pump protons across a membrane. Here, we use kinetic master equations to explore the energetic and kinetic control of proton pumping in CcO. We construct models consistent with thermodynamic principles, the structure of CcO, experimentally known proton affinities, and equilibrium constants of intermediate reactions. The resulting models are found to capture key properties of CcO, including the midpoint redox potentials of the metal centers and the electron transfer rates. We find that coarse-grained models with two proton sites and one electron site can pump one proton per electron against membrane potentials exceeding 100 mV. The high pumping efficiency of these models requires strong electrostatic couplings between the proton loading (pump) site and the electron site (heme a), and kinetic gating of the internal proton transfer. Gating is achieved by enhancing the rate of proton transfer from the conserved Glu-242 to the pump site on reduction of heme a, consistent with the predictions of the water-gated model of proton pumping. The model also accounts for the phenotype of D-channel mutations associated with loss of pumping but retained turnover. The fundamental mechanism identified here for the efficient conversion of chemical energy into an electrochemical potential should prove relevant also for other molecular machines and novel fuel-cell designs.

It is well known that there are various changes in the expression of hepatic and intestinal CYPs in mutant Nagase analbuminemic rats (NARs). It has been reported that the protein expression of hepatic CYP1A2 was increased, whereas that of hepatic CYP3A1 was not altered, and it was also found that the protein expression of the intestinal CYP1A subfamily significantly increased in NARs from our other study. In addition, in this study additional information about CYP changes in NARs was obtained; the protein expression of the hepatic CYP2D subfamily was not altered, but that of the intestinal CYP3A subfamily increased in NARs. Because omeprazole is metabolized via hepatic CYP1A1/2, 2D1, 3A1/2 in rats, it could be expected that the pharmacokinetics of omeprazole would be altered in NARs. After intravenous administration of omeprazole to NARs, the Cl(nr) was significantly faster than in the controls (110 versus 46.6 ml/min/kg), and this could be due to an increase in hepatic metabolism caused by a greater hepatic CYP1A2 level in addition to greater free fractions of the drug in NARs. After oral administration of omeprazole to NARs, the AUC was also significantly smaller (80.1% decrease) and F was decreased in NARs. This could be primarily due to increased hepatic and intestinal metabolism caused by greater hepatic CYP1A2 and intestinal CYP1A and 3A levels. In particular, the smaller F could mainly result from greater hepatic and intestinal first-pass effect in NARs than in the controls.

Nonalcoholic fatty liver is involved in the development of nonalcoholic steatohepatitis and chronic liver injury. Impairment of hepatic transsulfuration reactions is suggested to be critically linked with alcoholic liver injury, but its role in nonalcoholic fatty liver remains unknown. We examined the early changes in sulfur-amino acid metabolism and their implication in nonalcoholic fatty liver disease (NAFLD). Male rats were provided with a standard liquid diet or a high-fat liquid diet (HF) for 3 wk. An additional group of rats received the HF diet supplemented with betaine (1%). HF diet intake elevated hepatic triglyceride and serum tumor necrosis factor-alpha (TNFalpha) concentrations. Antioxidant capacity of liver cytosol against hydroxyl and peroxyl radicals was reduced significantly. Hepatic S-adenosylmethionine (SAM) and glutathione (GSH) decreased, but hypotaurine and taurine concentrations increased. Methionine adenosyltransferase (MAT) activity, not its concentration, was depressed, whereas both activity and concentration of cysteine dioxygenase and GSH S-transferase were elevated. Betaine supplementation of the HF diet inhibited hepatic fat accumulation and serum TNFalpha elevation. The decrease in cytosolic antioxidant capacity was also prevented. MAT activity and its concentration were induced significantly. Hepatic SAM and GSH increased and elevation of hypotaurine and taurine was depressed. The results indicate that the metabolism of S-containing substances is significantly disturbed by the HF diet, suggesting a causal role of impairment of hepatic transsulfuration reactions in NAFLD. Betaine supplementation protects the liver from nonalcoholic steatosis and oxidative stress most probably via its effects on the transsulfuration reactions.

Recent paramagnetic relaxation enhancement (PRE) studies on several weakly interacting protein complexes have unequivocally demonstrated the existence of transient encounter complexes. Here, we present a computational method to study protein-protein binding by creating equilibrium ensembles that include both specific and nonspecific protein complexes. In a joint analysis of simulation and experiment we explore the physical nature and underlying physicochemical characteristics of encounter complexes involving three protein-protein interactions of the bacterial phosphotransferase system. Replica exchange Monte Carlo simulations using a coarse-grained energy function recover the structures of the specific complexes and produce binding affinities in good agreement with experiment. Together with the specific complex, a relatively small number of distinct nonspecific complexes largely accounts for the measured PRE data. The combined relative population of the latter is less than approximately 10%. The binding interfaces of the specific and nonspecific complexes differ primarily in size but exhibit similar amino acid compositions. We find that the overall funnel-shaped energy landscape of complex formation is dominated by the specific complex, a small number of structured nonspecific complexes, and a diffuse cloud of loosely bound complexes connecting the specific and nonspecific binding sites with each other and the unbound state. Nonspecific complexes may not only accelerate the binding kinetics by enhancing the rate of success of random diffusional encounters but also play a role in protein function as alternative binding modes.

It has been reported that omeprazole is mainly metabolized via hepatic cytochrome P450 (CYP) 1A1/2, CYP2D1 and CYP3A1/2 in male Sprague-Dawley rats, and the expression of hepatic CYP3A1 is increased in male Sprague-Dawley rats with acute renal failure induced by uranyl nitrate (U-ARF rats). Thus, the metabolism of omeprazole would be expected to increase in U-ARF rats. After intravenous administration of omeprazole (20 mgkg(-1)) to U-ARF rats, the area under the plasma concentration-time curve from time zero to infinity (AUC) was significantly reduced (371 vs 494 microg min mL(-1)), possibly due to the significantly faster non-renal clearance (56.6 vs 41.2 mL min(-1) kg(-1)) compared with control rats. This could have been due to increased expression of hepatic CYP3A1 in U-ARF rats. After oral administration of omeprazole (40 mgkg(-1)) to U-ARF rats, the AUC was also significantly reduced (89.3 vs 235 microg min mL(-1)) compared with control rats. The AUC difference after oral administration (62.0% decrease) was greater than that after intravenous administration (24.9% decrease). This may have been primarily due to increased intestinal metabolism of omeprazole caused by increased expression of intestinal CYP1A and 3A subfamilies in U-ARF rats, in addition to increased hepatic metabolism.

We develop coarse-grained models and effective energy functions for simulating thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (K(d) >1 microM) and apply them to binding of Vps27 to membrane-tethered ubiquitin. Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Hückel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. In simulations of membrane-attached protein complexes, interactions between amino acids and the membrane are described by residue-dependent short-range potentials and long-range electrostatics. We parameterize the energy functions by fitting the osmotic second virial coefficient of lysozyme and the binding affinity of the ubiquitin-CUE complex. For validation, extensive replica-exchange Monte Carlo simulations are performed of various protein complexes. Binding affinities for these complexes are in good agreement with the experimental data. The simulated structures are clustered on the basis of distance matrices between two proteins and ranked according to cluster population. In approximately 70% of the complexes, the distance root-mean-square is less than 5 A from the experimental structures. In approximately 90% of the complexes, the binding interfaces on both proteins are predicted correctly, and in all other cases at least one interface is correct. Transient and nonspecifically bound structures are also observed. With the validated model, we simulate the interaction between the Vps27 multiprotein complex and a membrane-tethered ubiquitin. Ubiquitin is found to bind preferentially to the two UIM domains of Vps27, but transient interactions between ubiquitin and the VHS and FYVE domains are observed as well. These specific and nonspecific interactions are found to be positively cooperative, resulting in a substantial enhancement of the overall binding affinity beyond the approximately 300 microM of the specific domains. We also find that the interactions between ubiquitin and Vps27 are highly dynamic, with conformational rearrangements enabling binding of Vps27 to diverse targets as part of the multivesicular-body protein-sorting pathway.

Injury to liver, resulting in loss of its normal physiological/biochemical functions, may adversely affect a secondary organ. We examined the response of the liver and kidney to chemical substances that require metabolic activation for their toxicities in mice with a preceding liver injury. Carbon tetrachloride treatment 24 h prior to a challenging dose of carbon tetrachloride or acetaminophen decreased the resulting hepatotoxicity both in male and female mice as determined by histopathological examination and increases in serum enzyme activities. In contrast, the renal toxicity of the challenging toxicants was elevated markedly in male, but not in female mice. Partial hepatectomy also induced similar changes in the hepatotoxicity and nephrotoxicity of a challenging toxicant, suggesting that the contrasting response of male liver and kidney was associated with the reduction of the hepatic metabolizing capacity. Carbon tetrachloride pretreatment or partial hepatectomy decreased the hepatic xenobiotic-metabolizing enzyme activities in both sexes but elevated the renal p-nitrophenol hydroxylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase activities significantly only in male mice. Increases in Cyp2e1 and Cyp2b expression were also evident in male kidney. Castration of males or testosterone administration to females diminished the sex-related differences in the renal response to an acute liver injury. The results indicate that reduction of the hepatic metabolizing capacity induced by liver injury may render secondary target organs susceptible to chemical substances activated in these organs. This effect may be sex-specific. It is also suggested that an integrated approach should be taken for proper assessment of chemical hazards.

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, pumps protons across the inner mitochondrial membrane against an opposing electrochemical gradient by reducing oxygen to water. To explore the fundamental mechanisms of such redox-coupled proton pumps, we develop kinetic models at the single-molecule level consistent with basic physical principles. We demonstrate that pumping against potentials >150 mV can be achieved purely through electrostatic couplings, given an asymmetric arrangement of charge centers; however, nonlinear gates are essential for highly efficient real enzymes. The fundamental requirements for proton pumping identified here highlight a possible evolutionary origin of cytochrome c oxidase pumping. The general design principles are relevant also for other molecular machines and suggest future applications in biology-inspired fuel cells.

The neuroprotective effect of mitochondrial isocitrate dehydrogenase (IDPm), an enzyme involved in the reduction of NADP(+) to NADPH and the supply of glutathione (GSH) in mitochondria, was examined using SH-SY5Y cells overexpressing IDPm (S1). S1 cells showed higher NADPH and GSH levels than vector transfectant (V) cells and were more resistant to staurosporine-induced cell death than controls. Staurosporine-induced cytochrome c release, caspase-3 activation, and production of reactive oxygen species (ROS) were significantly attenuated in S1 cells as compared to V cells and reduced by antioxidants, trolox and GSH-ethyl ester (GSH-EE). Staurosporine-induced the release of Mcl-1 from mitochondria that formed a complex with Bim. Mcl-1 was then cleaved to a shortened form in a caspase-3 dependent manner; its release was attenuated far more in S1 than in V cells after staurosporine treatment. Finally, the staurosporine-induced decrease in mitochondrial membrane potential (Deltapsi(m)) was correlated with the time of mitochondrial Mcl-1 release; the loss of Deltapsi(m) was attenuated significantly in S1 cells as compared to that in V cells. These results suggest that the neuroprotective effect of IDPm may result from increases in NADPH and GSH levels in the mitochondria. This, in turn, inhibits mitochondrial ROS production after cytochrome c release, which seems to be mediated through Mcl-1 release.

Antioxidant enzymes including superoxide dismutase (SOD) may play a role in the mechanism by which cells counteract the deleterious effects of reactive oxygen species (ROS) after spinal cord injury (SCI). Cu/Zn and MnSOD are especially potent scavengers of superoxide anion and likely serve important cytoprotective roles against cellular damage. We investigated expression of SOD after SCI to address its role during the early stages of injury. MnSOD activity was increased 4 h after SCI and persisted at elevated levels up to 24-48 h; by contrast, Cu/ZnSOD activity was not changed. RT-PCR and Western blot analyses showed increased levels of MnSOD mRNA and protein, respectively, by 4 h and reached maximum levels by 24-48 h. Double immunostaining revealed that MnSOD protein was localized within neurons and oligodendrocytes. Tumor necrosis factor-alpha (TNF-alpha) was administered locally into uninjured spinal cords to examine potential mechanisms for MnSOD induction after injury. TNF-alpha administered exogenously increased MnSOD expression in uninjured spinal cords. Western blot and immunostaining also revealed that a transcription factor, NF-kappaB, was activated and translocated into the nuclei of neurons and oligodendrocytes. By contrast, administration of neutralizing antibody against TNF-alpha into injured spinal cords attenuated the increase in MnSOD expression and activation of NF-kappaB. Double immunostaining revealed that MnSOD was co-localized with NF-kappaB in neurons and oligodendrocytes after SCI. These results suggest that TNF-alpha may be an inducer of NF-kappaB activation and MnSOD expression after SCI and that MnSOD expression induced by TNF-alpha is likely mediated through activation of NF-kappaB.

We showed previously that, after spinal cord injury (SCI), tumor necrosis factor-alpha (TNF-alpha) may serve as an external signal, initiating apoptosis in neurons and oligodendrocytes. To further characterize the apoptotic cascade initiated by TNF-alpha after SCI, we examined the expression of TNF-alpha, inducible nitric oxide (NO) synthase (iNOS), and the level of NO after SCI. Western blots and reverse transcription polymerase chain reactions showed an early upregulation of TNF-alpha after injury. A peak TNF-alpha expression was observed within 1 h of injury. By 4 h after injury, the expression of iNOS and the level of NO were markedly increased in the injured spinal cord. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive cells were also first observed in the lesioned area 4 h after SCI. The largest number of TUNEL-positive cells was observed between 24-48 h after SCI. Injecting a neutralizing antibody against TNF-alpha into the lesion site after injury significantly reduced the expression of iNOS, the level of NO and the number of TUNEL-positive cells in the injured spinal cord. Injecting the NOS inhibitors, N(G)-monomethyl-L-arginine monoacetate and S-methylisothiourea sulfate, or an NO scavenger, carboxy-PTIO, into the lesion site also significantly reduced the level of NO and the degree of DNA laddering in the injured spinal cord. These data suggest that after SCI, apoptosis induced by TNF-alpha may be mediated in part by NO via upregulation of iNOS, induced in response to TNF-alpha.

Alterations in the hepatic metabolism of S-amino acids were examined in male rats injected with a single dose of ethanol (3 g/kg, i.p.). The hepatic concentrations of methionine and S-adenosylhomocysteine (SAH) were increased, but S-adenosylmethionine (SAM), cysteine, and glutathione (GSH) decreased rapidly following ethanol administration. The activities of methionine adenosyltransferase (MAT), cystathionine beta-synthase (CbetaS) and cystathionine gamma-lyase (CgammaL) were all inhibited. Gamma-glutamylcysteine synthetase (GCS) activity was increased from t = 8 hr, but hepatic glutathione (GSH) level did not return to control for 48 hr. Both hepatic hypotaurine and taurine levels were increased immediately, which were reduced to below control from t = 18 hr. Changes in the serum concentration of taurine were consistent with results observed in the liver. Cysteine dioxygenase (CDO) activity was increased rapidly, but declined from t = 24 hr. The results indicate that an acute dose of ethanol induces significant alterations in the metabolism of S-amino acids in the liver. Ethanol depresses the cysteine availability for GSH synthesis not only by inhibiting the transsulfuration reactions but also by enhancing its irreversible catabolism to taurine via hypotaurine. The physiological significance of this finding is discussed.