Docking-based long timescale simulation of cell-size protein systems at atomic resolution.

Computational methodologies are increasingly addressing modeling of the whole cell at the molecular level. Proteins and their interactions are the key component of cellular processes. Techniques for modeling protein interactions, thus far, have included protein docking and molecular simulation. The latter approaches account for the dynamics of the interactions but are relatively slow, if carried out at all-atom resolution, or are significantly coarse grained. Protein docking algorithms are far more efficient in sampling spatial coordinates. However, they do not account for the kinetics of the association (i.e., they do not involve the time coordinate). Our proof-of-concept study bridges the two modeling approaches, developing an approach that can reach unprecedented simulation timescales at all-atom resolution. The global intermolecular energy landscape of a large system of proteins was mapped by the pairwise fast Fourier transform docking and sampled in space and time by Monte Carlo simulations. The simulation protocol was parametrized on existing data and validated on a number of observations from experiments and molecular dynamics simulations. The simulation protocol performed consistently across very different systems of proteins at different protein concentrations. It recapitulated data on the previously observed protein diffusion rates and aggregation. The speed of calculation allows reaching second-long trajectories of protein systems that approach the size of the cells, at atomic resolution.

Strength in numbers: effect of protein crowding on the shape of cell membranes.

Continuous reshaping of the plasma membrane into pleomorphic shapes is critical for a plethora of cellular functions. How the cell carries out this enigmatic control of membrane remodeling has remained an active research field for decades and several molecular and biophysical mechanisms have shown to be involved in overcoming the energy barrier associated with membrane bending. The reported mechanisms behind membrane bending have been largely concerned with structural protein features, however, in the last decade, reports on the ability of densely packed proteins to bend membranes by protein-protein crowding, have challenged prevailing mechanistic views. Crowding has now been shown to generate spontaneous vesicle formation and tubular morphologies on cell- and model membranes, demonstrating crowding as a relevant player involved in the bending of membranes. Still, current research is largely based on unnatural overexpression of proteins in non-native domains, and together with efforts in modeling, this has led to questioning the in vivo impact of crowding. In this review, we examine this previously overlooked mechanism by summarizing recent advances in the understanding of protein-protein crowding and its prevalence in cellular membrane-shaping processes.

Nanobody-mediated macromolecular crowding induces membrane fission and remodeling in the African trypanosome.

The dense variant surface glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host's antibody response. To explore the epitope space of VSGs, we generate anti-VSG nanobodies and combine high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody causes rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endocytosis and exocytosis are mechanistically linked to Trypanosoma brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrate that this loss of motility is accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells.

BiFC Method Based on Intraorganellar Protein Crowding Detects Oleate-Dependent Peroxisomal Targeting of Pichia pastoris Malate Dehydrogenase.

The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.

Protein crowding mediates membrane remodeling in upstream ESCRT-induced formation of intraluminal vesicles.

As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs). Whereas ESCRT-III/VPS4 have an established function in ILV formation, the role of upstream ESCRTs (0 to II) in membrane shape remodeling is not understood. Combining experimental measurements and electron microscopy analysis of ESCRT-III-depleted cells with a mathematical model, we show that upstream ESCRT-induced alteration of the Gaussian bending rigidity and their crowding in concert with the transmembrane cargo on the membrane induce membrane deformation and facilitate ILV formation: Upstream ESCRT-driven budding does not require ATP consumption as only a small energy barrier needs to be overcome. Our model predicts that ESCRTs do not become part of the ILV, but localize with a high density at the membrane neck, where the steep decline in the Gaussian curvature likely triggers ESCRT-III/VPS4 assembly to enable neck constriction and scission.

Hydrogels from serum albumin in a molten globule-like state.

We demonstrate that a molten globule-like (MG) state of a protein, usually described as a compact yet non-folded conformation that is only present in a narrow and delicate parameter range, is preserved in the high concentration environment of the protein hydrogel. We reveal mainly by means of electron paramagnetic resonance (EPR) spectroscopy that bovine serum albumin (BSA) retains the known basic MG state after a hydrogel has been formed from 20 wt% precursor solutions. At pH values of ~11.4, BSA hydrogels made from MG-BSA remain stable for weeks, while gels formed at slightly different (~0.2) pH units above and below the MG-state value dissolve into viscous solutions. On the contrary, when hydrophobic screening agents are added such as amphiphilic, EPR-active stearic acid derivatives (16-DOXYL-stearic acid, 16-DSA), the MG-state based hydrogel is the least long-lived, as the hydrophobic interaction of delicately exposed hydrophobic patches of BSA molecules is screened by the amphiphilic molecules. These bio- and polymer-physically unexpected findings may lead to new bio-compatible MG-based hydrogels that display novel properties in comparison to conventional gels.

Dynamic cluster formation determines viscosity and diffusion in dense protein solutions.

We develop a detailed description of protein translational and rotational diffusion in concentrated solution on the basis of all-atom molecular dynamics simulations in explicit solvent. Our systems contain up to 540 fully flexible proteins with 3.6 million atoms. In concentrated protein solutions (100 mg/mL and higher), the proteins ubiquitin and lysozyme, as well as the protein domains third IgG-binding domain of protein G and villin headpiece, diffuse not as isolated particles, but as members of transient clusters between which they constantly exchange. A dynamic cluster model nearly quantitatively explains the increase in viscosity and the decrease in protein diffusivity with protein volume fraction, which both exceed the predictions from widely used colloid models. The Stokes-Einstein relations for translational and rotational diffusion remain valid, but the effective hydrodynamic radius grows linearly with protein volume fraction. This increase follows the observed increase in cluster size and explains the more dramatic slowdown of protein rotation compared with translation. Baxter's sticky-sphere model of colloidal suspensions captures the concentration dependence of cluster size, viscosity, and rotational and translational diffusion. The consistency between simulations and experiments for a diverse set of soluble globular proteins indicates that the cluster model applies broadly to concentrated protein solutions, with equilibrium dissociation constants for nonspecific protein-protein binding in the Kd ≈ 10-mM regime.

Modeling Membrane Curvature Generation due to Membrane⁻Protein Interactions.

To alter and adjust the shape of the plasma membrane, cells harness various mechanisms of curvature generation. Many of these curvature generation mechanisms rely on the interactions between peripheral membrane proteins, integral membrane proteins, and lipids in the bilayer membrane. Mathematical and computational modeling of membrane curvature generation has provided great insights into the physics underlying these processes. However, one of the challenges in modeling these processes is identifying the suitable constitutive relationships that describe the membrane free energy including protein distribution and curvature generation capability. Here, we review some of the commonly used continuum elastic membrane models that have been developed for this purpose and discuss their applications. Finally, we address some fundamental challenges that future theoretical methods need to overcome to push the boundaries of current model applications.

Intronic miRNA mediated gene expression regulation controls protein crowding inside the cell.

Gene regulatory effects of microRNAs at a posttranscriptional level have been established over the last decade. In this study, we analyze the interaction networks of mRNA translation regulation through intronic miRNA, under various tissue-specific cellular contexts, taking into account the thermodynamic affinity, chemical kinetics, co-localization, concentration levels, network parameters and the presence of competitive interactors. This database, and analysis has been made available through an open-access web-server, miRiam, to promote further exploration. Here we report that expression of genes involved in Apoptosis Processes, Immune System Processes, Translation Regulator Activities, and Molecular Transport Activities within the cell are predominately regulated by miRNA mediation. Our findings further indicate that this regulatory effect has a profound effect in controlling protein crowding inside the cell. A miRNA mediated gene expression regulation serves as a temporal regulator, allowing the cellular machinery to temporarily 'pause' the translation of mRNA, indicating that the miRNA-mRNA interactions may be important for governing the optimal usage of cell volume.

Structure Versus Stochasticity-The Role of Molecular Crowding and Intrinsic Disorder in Membrane Fission.

Cellular membranes must undergo remodeling to facilitate critical functions including membrane trafficking, organelle biogenesis, and cell division. An essential step in membrane remodeling is membrane fission, in which an initially continuous membrane surface is divided into multiple, separate compartments. The established view has been that membrane fission requires proteins with conserved structural features such as helical scaffolds, hydrophobic insertions, and polymerized assemblies. In this review, we discuss these structure-based fission mechanisms and highlight recent findings from several groups that support an alternative, structure-independent mechanism of membrane fission. This mechanism relies on lateral collisions among crowded, membrane-bound proteins to generate sufficient steric pressure to drive membrane vesiculation. As a stochastic process, this mechanism contrasts with the paradigm that deterministic protein structures are required to drive fission, raising the prospect that many more proteins may participate in fission than previously thought. Paradoxically, our recent work suggests that intrinsically disordered domains may be among the most potent drivers of membrane fission, owing to their large hydrodynamic radii and substantial chain entropy. This stochastic view of fission also suggests new roles for the structure-based fission proteins. Specifically, we hypothesize that in addition to driving fission directly, the canonical fission machines may facilitate the enrichment and organization of bulky disordered protein domains in order to promote membrane fission by locally amplifying protein crowding.

Crowd-Sourcing of Membrane Fission: How crowding of non-specialized membrane-bound proteins contributes to cellular membrane fission.

Fission of cellular membranes is ubiquitous and essential for life. Complex protein machineries, such as the dynamin and ESCRT spirals, have evolved to mediate membrane fission during diverse cellular processes, for example, vesicle budding. A new study suggests that non-specialized membrane-bound proteins can induce membrane fission through mass action due to protein crowding. Because up to 2/3 of the mass of cellular membranes is contributed by proteins, membrane protein crowding is an important physiological parameter. Considering the complexity of membrane shape transitions during a fission reaction, spatial and temporal variability in protein distribution, and the abundance of intrinsically disordered regions in proteins on an invaginating membrane, protein crowding can have diverse consequences for fission in the cell. The question is, how and to what extent this mechanism combines with the action of dedicated fission machineries.

Catanionic Coacervate Droplets as a Surfactant-Based Membrane-Free Protocell Model.

We report on the formation of surfactant-based complex catanionic coacervate droplets in mixtures of decanoic acid and cetylpyridinium chloride or cetyltrimethylammonium bromide. We show that coacervation occurs over a broad range of composition, pH, and ionic strength. The catanionic coacervates consist of elongated micelles, sequester a wide range of solutes including water-soluble organic dyes, polysaccharides, proteins, enzymes, and DNA, and can be structurally stabilized by sodium alginate or gelatin-based hydrogelation. These results suggest that catanionic coacervates could be exploited as a novel surfactant-based membrane-free protocell model.

Why Do Fast-Growing Bacteria Enter Overflow Metabolism? Testing the Membrane Real Estate Hypothesis.

Bacteria and other cells show a puzzling behavior. At high growth rates, E. coli switch from respiration (which is ATP-efficient) to using fermentation for additional ATP (which is inefficient). This overflow metabolism results in a several-fold decrease in ATP produced per glucose molecule provided as food. By integrating diverse types of experimental data into a simple biophysical model, we give evidence that this onset is the result of the membrane real estate hypothesis: Fast growth drives cells to be bigger, reducing their surface-to-volume ratios. This decreases the membrane area available for respiratory proteins despite growing demand, causing increased crowding. Only when respiratory proteins reach their crowding limit does the cell activate fermentation, since fermentation allows faster ATP production per unit membrane area. Surface limitation thus creates a Pareto trade-off between membrane efficiency and ATP yield that links metabolic choice to the size and shape of a bacterial cell. By exploring the predictions that emerge from this trade-off, we show how consideration of molecular structures, energetics, rates, and equilibria can provide important insight into cellular behavior.

Dilation of fusion pores by crowding of SNARE proteins.

Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, 'flipped' t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion).

Dramatic influence of patchy attractions on short-time protein diffusion under crowded conditions.

In the dense and crowded environment of the cell cytoplasm, an individual protein feels the presence of and interacts with all surrounding proteins. While we expect this to strongly influence the short-time diffusion coefficient Ds of proteins on length scales comparable to the nearest-neighbor distance, this quantity is difficult to assess experimentally. We demonstrate that quantitative information about Ds can be obtained from quasi-elastic neutron scattering experiments using the neutron spin echo technique. We choose two well-characterized and highly stable eye lens proteins, bovine α-crystallin and γB-crystallin, and measure their diffusion at concentrations comparable to those present in the eye lens. While diffusion slows down with increasing concentration for both proteins, we find marked variations that are directly linked to subtle differences in their interaction potentials. A comparison with computer simulations shows that anisotropic and patchy interactions play an essential role in determining the local short-time dynamics. Hence, our study clearly demonstrates the enormous effect that weak attractions can have on the short-time diffusion of proteins at concentrations comparable to those in the cellular cytosol.

Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity.

Gene dosage toxicity (GDT) is an important factor that determines optimal levels of protein abundances, yet its molecular underpinnings remain unknown. Here, we demonstrate that overexpression of DHFR in E. coli causes a toxic metabolic imbalance triggered by interactions with several functionally related enzymes. Though deleterious in the overexpression regime, surprisingly, these interactions are beneficial at physiological concentrations, implying their functional significance in vivo. Moreover, we found that overexpression of orthologous DHFR proteins had minimal effect on all levels of cellular organization - molecular, systems, and phenotypic, in sharp contrast to E. coli DHFR. Dramatic difference of GDT between 'E. coli's self' and 'foreign' proteins suggests the crucial role of evolutionary selection in shaping protein-protein interaction (PPI) networks at the whole proteome level. This study shows how protein overexpression perturbs a dynamic metabolon of weak yet potentially functional PPI, with consequences for the metabolic state of cells and their fitness.

Membrane bending by protein crowding is affected by protein lateral confinement.

Crowding of asymmetrically-distributed membrane proteins has been recently recognized as an important factor in remodeling of biological membranes, for example during transport vesicle formation. In this paper, we theoretically analyze the effect of protein crowding on membrane bending and examine its dependence on protein size, shape, transmembrane asymmetry and lateral confinement. We consider three scenarios of protein lateral organization, which are highly relevant for cellular membranes in general: freely diffusing membrane proteins without lateral confinement, the presence of a diffusion barrier and interactions with a vesicular coat. We show that protein crowding affects vesicle formation even if the proteins are distributed symmetrically across the membrane and that this effect depends significantly on lateral confinement. The largest crowding effect is predicted for the proteins that are confined to the forming vesicle by a diffusion barrier. We calculate the bending properties of a crowded membrane and find that its spontaneous curvature depends primarily on the degree of transmembrane asymmetry, and its effective bending modulus on the type of lateral confinement. Using the example of COPII vesicle formation from the endoplasmic reticulum, we analyze the energetic cost of vesicle formation. The results provide a novel insight into the effects of lateral and transmembrane organization of membrane proteins, and can guide data interpretation and future experimental approaches.

Non-Brownian diffusion in lipid membranes: Experiments and simulations.

The dynamics of constituents and the surface response of cellular membranes-also in connection to the binding of various particles and macromolecules to the membrane-are still a matter of controversy in the membrane biophysics community, particularly with respect to crowded membranes of living biological cells. We here put into perspective recent single particle tracking experiments in the plasma membranes of living cells and supercomputing studies of lipid bilayer model membranes with and without protein crowding. Special emphasis is put on the observation of anomalous, non-Brownian diffusion of both lipid molecules and proteins embedded in the lipid bilayer. While single component, pure lipid bilayers in simulations exhibit only transient anomalous diffusion of lipid molecules on nanosecond time scales, the persistence of anomalous diffusion becomes significantly longer ranged on the addition of disorder-through the addition of cholesterol or proteins-and on passing of the membrane lipids to the gel phase. Concurrently, experiments demonstrate the anomalous diffusion of membrane embedded proteins up to macroscopic time scales in the minute time range. Particular emphasis will be put on the physical character of the anomalous diffusion, in particular, the occurrence of ageing observed in the experiments-the effective diffusivity of the measured particles is a decreasing function of time. Moreover, we present results for the time dependent local scaling exponent of the mean squared displacement of the monitored particles. Recent results finding deviations from the commonly assumed Gaussian diffusion patterns in protein crowded membranes are reported. The properties of the displacement autocorrelation function of the lipid molecules are discussed in the light of their appropriate physical anomalous diffusion models, both for non-crowded and crowded membranes. In the last part of this review we address the upcoming field of membrane distortion by elongated membrane-binding particles. We discuss how membrane compartmentalisation and the particle-membrane binding energy may impact the dynamics and response of lipid membranes. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Conformational changes in calcium-sensor proteins under molecular crowding conditions.

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca(2+) sensors, which undergo conformational changes in response to oscillating intracellular Ca(2+) concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca(2+)-binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase-activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca(2+)-sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca(2+) conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size-exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein-water interface. Our study provides insight into how rather small signaling proteins that have very similar three-dimensional folding patterns differ in their Ca(2+)-occupied functional state under crowding conditions.

Membrane bending: the power of protein imbalance.

Many cellular processes require membrane deformation, which is driven by specialized protein machinery and can often be recapitulated using pure lipid bilayers. However, biological membranes contain a large amount of embedded proteins. Recent research suggests that membrane-bound proteins with asymmetric distribution of mass across the bilayer can influence membrane bending in a nonspecific manner due to molecular crowding. This mechanism is physical in nature and arises from collisions between such 'mushroom-shaped' proteins. It can either facilitate or impede the action of protein coats, for example COPII, during vesicle budding. We describe the physics of how molecular crowding can influence membrane bending and discuss the implications for other cellular processes, such as sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and production of intraluminal vesicles.