No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Protein-Protein Association in Polymer Solutions: From Dilute to Semidilute to Concentrated
Abstract
In a typical cell, proteins function in the crowded cytoplasmic environment where 30% of the space is occupied by macromolecules of varying size and nature. This environment may be simulated in vitro using synthetic polymers. Here, we followed the association and diffusion rates of TEM1-beta-lactamase (TEM) and the beta-lactamase inhibitor protein (BLIP) in the presence of crowding agents of varying molecular mass, from monomers (ethylene glycol, glycerol, or sucrose) to polymeric agents such as different polyethylene glycols (PEGs, 0.2-8 kDa) and Ficoll. An inverse linear relation was found between translational diffusion of the proteins and viscosity in all solutions tested, in accordance with the Stokes-Einstein (SE) relation. Conversely, no simple relation was found between either rotational diffusion rates or association rates (k(on)) and viscosity. To assess the translational diffusion-independent steps along the association pathway, we introduced a new factor, alpha, which corrects the relative change in k(on) by the relative change in solution viscosity, thus measuring the deviations of the association rates from SE behavior. We found that these deviations were related to the three regimes of polymer solutions: dilute, semidilute, and concentrated. In the dilute regime PEGs interfere with TEM-BLIP association by introducing a repulsive force due to solvophobic preferential hydration, which results in slower association than predicted by the SE relation. Crossing over from the dilute to the semidilute regime results in positive deviations from SE behavior, i.e., relatively faster association rates. These can be attributed to the depletion interaction, which results in an effective attraction between the two proteins, winning over the repulsive force. In the concentrated regime, PEGs again dramatically slow down the association between TEM and BLIP, an effect that does not depend on the physical dimensions of PEGs, but rather on their mass concentration. This is probably a manifestation of the monomer-like repulsive depletion effect known to occur in concentrated polymer solutions. As a transition from moderate to high crowding agent concentration can occur in the cellular milieu, this behavior may modulate protein association in vivo, thereby modulating biological function.
... A semidilute PEG solution, where the polymer molecules are highly overlapping, behaves more like a solution of monomer segments than that of individual polymer molecules. 50,51 While the monomer segments contribute more significantly to osmotic pressure than the individual polymer molecules, 52 they are not effective in inducing depletion interaction. 50,51 For this reason, eq 2 underestimates the depletion interaction (Δv) at high PEG concentrations in Figure 4A. ...
... 50,51 While the monomer segments contribute more significantly to osmotic pressure than the individual polymer molecules, 52 they are not effective in inducing depletion interaction. 50,51 For this reason, eq 2 underestimates the depletion interaction (Δv) at high PEG concentrations in Figure 4A. In the new model (eq 5), the empirical exponent of the PEG concentration term (0.611) is smaller than 1, which is consistent with that the PEG-induced depletion interaction grows slower than the PEG concentration due to overlapping of the PEG molecules in the semidilute region. ...
... In the new model (eq 5), the empirical exponent of the PEG concentration term (0.611) is smaller than 1, which is consistent with that the PEG-induced depletion interaction grows slower than the PEG concentration due to overlapping of the PEG molecules in the semidilute region. 50,51 Therefore, eq 5 provides more accurate physical insights than the old model into the effects of depletion interaction on PEG-induced LLPS. ...
... Next, taking cue from the quinary interactions gaining ground in the 'crowding' community, the proteins with their patches of hydrophilic and hydrophobic residues are geared towards exerting appreciable non-specific interactions [46][47][48][49]. Moreover, while polymer based crowders (as the crowders except BSA, used in this study are), have the intrinsic property of entanglement [23,[50][51][52], the structured protein based crowder does not. On the contrary, the latter can aggregate (not entangle) giving rise to a different type of caging effect. ...
... Indeed, after 150 g/L, there was no further increase in the ThT fluorescence, that is, the aggregated ensemble of BSA remained constant, thereby also not bringing about further changes in the protein dynamics. Amongst the synthetic crowders, which have also been referred to as uniform crowding agents [52][53][54][55][56], Ficoll 70 induces the maximum retardation in protein dynamics, with Dextran 40 exerting the least influence. While the variation amongst these macromolecular crowders can be primarily attributed to their difference in morphology, recent evidence provides greater details about the potential origins of the observed differences [57]. ...
Understanding the effect of mixed crowding is of prime importance with regards to addressing the manner in which the crowded cellular interior influences the structure, function and dynamics of proteins. In this study we have used the internal dynamics of the multidomain serum protein, bovine serum albumin (BSA), labeled with the solvation probe 7-diethylamino-3-4-maleimidophenyl-4-methylcoumarin (CPM), as a sensor for binary crowding mixtures. Both synthetic (Ficoll 70, Dextran 70, Dextran 40 & PEG 8000) and protein based (unlabeled BSA) crowders have been used. The ‘BSA+Ficoll 70’ mixture had the maximum retardation effect on the protein dynamics with the average solvation time being more than that of the sum of the individual crowding agents. On the contrary, all other binary mixtures had the reverse effect with the excluded volume induced in their presence being lesser than that of the component crowders taken together. Further support was obtained from the overall protein dynamics based on the acrylamide quenching of emission of the tryptophan residues of BSA. Our results have been discussed in the light of the compatibility and lack in thereof, of the individual components of the mixtures, an aspect that has been further supported by excess viscosity (ηE) estimations based on our time resolved anisotropy data. Till date, crowding induced excluded volume has been sensed mostly through diffusion-based experiments or FRET based sensors. As shown in this study, internal protein dynamics can also add another dimension to the manner in which the degree of macromolecular congestion can be sensed, with the possibility of mapping site-specific response along the polypeptide backbone.
... Macromolecular crowders have conventionally been known to give rise to hard-core repulsions arising from the mutual impenetrability of molecules, this being commonly referred to as the excluded volume effect [8][9][10]. Crowding effects on enzymes in solution have been described by changes in effective concentration of the substrate and enzyme [11,12], slowed down diffusion of enzyme and/or substrate [12,13], interactions between crowder and substrate [14], and proteinprotein association in polymer solutions [15,16]. Even after many years of research, a number of facets, like the chemical interactions between the crowders and the protein, non-specific protein-polymer and substrate-polymer interactions [16][17][18], protein conformational fluctuations due to increased viscosity or steric repulsions, decreased diffusion of enzyme and/or substrate [12,13,19,20], and their effects on catalytic activity, remain less understood. ...
... Crowding effects on enzymes in solution have been described by changes in effective concentration of the substrate and enzyme [11,12], slowed down diffusion of enzyme and/or substrate [12,13], interactions between crowder and substrate [14], and proteinprotein association in polymer solutions [15,16]. Even after many years of research, a number of facets, like the chemical interactions between the crowders and the protein, non-specific protein-polymer and substrate-polymer interactions [16][17][18], protein conformational fluctuations due to increased viscosity or steric repulsions, decreased diffusion of enzyme and/or substrate [12,13,19,20], and their effects on catalytic activity, remain less understood. Indeed, it has been challenging to predict the effect that these crowders can have on enzymes, with the activity of the latter having been shown to either increase [21,22], decrease [11,12,22] or remain the same [12,22,23], depending on the crowder and/or the enzyme under investigation [24]. ...
Macromolecular crowding, inside the physiological interior, modulates the energy landscape of biological macromolecules in multiple ways. Amongst these, enzymes occupy a special place and hence understanding the function of the same in the crowded interior is of utmost importance. In this study, we have investigated the manner in which the multidomain enzyme, AK3L1 (PDB ID: 1ZD8), an isoform of adenylate kinase, has its features affected in presence of commonly used crowders (PEG 8, Dextran 40, Dextran 70, and Ficoll 70). Michaelis Menten plots reveal that the crowders in general enhance the activity of the enzyme, with the Km and Vmax values showing significant variations. Ficoll 70, induced the maximum activity for AK3L1 at 100 g/L, beyond which the activity reduced. Ensemble FRET studies were performed to provide insights into the relative domain (LID and CORE) displacements in presence of the crowders. Solvation studies reveal that the protein matrix surrounding the probe CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin) gets restricted in presence of the crowders, with Ficoll 70 providing the maximum rigidity, the same being linked to the decrease in the activity of the enzyme. Through our multipronged approach, we have observed a distinct correlation between domain displacement, enzyme activity and associated dynamics. Thus, keeping in mind the complex nature of enzyme activity and the surrounding bath of dense soup that the biological entity remains immersed in, indeed more such approaches need to be undertaken to have a better grasp of the “enzymes in the crowd”.
... Macromolecular crowding describes the effect of proteins or complexes (e.g., microtubules, proteasomes) that exclude other small molecules from the space that they occupy. The presence of PEG of ∼2.5 nm or ∼20 nm radius leads to reduced association equilibria between TEM1-β-lactamase and β-lactamase inhibitor protein in vitro compared to solutions lacking PEG (Kozer et al., 2007;Zhou et al., 2008). This reduced association is thought to arise from the reduced rate of diffusion of the reactants (Kozer et al., 2007), perhaps due to reduced available solution space. ...
... The presence of PEG of ∼2.5 nm or ∼20 nm radius leads to reduced association equilibria between TEM1-β-lactamase and β-lactamase inhibitor protein in vitro compared to solutions lacking PEG (Kozer et al., 2007;Zhou et al., 2008). This reduced association is thought to arise from the reduced rate of diffusion of the reactants (Kozer et al., 2007), perhaps due to reduced available solution space. The relative decrease in the diffusive movement for any diffusing molecule in the cytosol as compared to water arising both from macromolecular crowding and the viscosity of the cytosol is termed as microscopic viscosity (Lavalette et al., 1999). ...
High concentration of cytoskeletal filaments, organelles, and proteins along with the space constraints due to the axon’s narrow geometry lead inevitably to intracellular physical crowding along the axon of a neuron. Local cargo movement is essential for maintaining steady cargo transport in the axon, and this may be impeded by physical crowding. Molecular motors that mediate active transport share movement mechanisms that allow them to bypass physical crowding present on microtubule tracks. Many neurodegenerative diseases, irrespective of how they are initiated, show increased physical crowding owing to the greater number of stalled organelles and structural changes associated with the cytoskeleton. Increased physical crowding may be a significant factor in slowing cargo transport to synapses, contributing to disease progression and culminating in the dying back of the neuronal process. This review explores the idea that physical crowding can impede cargo movement along the neuronal process. We examine the sources of physical crowding and strategies used by molecular motors that might enable cargo to circumvent physically crowded locations. Finally, we describe sub-cellular changes in neurodegenerative diseases that may alter physical crowding and discuss the implications of such changes on cargo movement.
... Mixtures of polyelectrolytes and like-charged colloidal particles in an aqueous salt solution are ubiquitous in both industrial applications [12,13] and biological systems [14,15]. A typical example is the high concentration of various charged macromolecules and proteins in living cells, leading to macromolecular crowding phenomena [15]. ...
Depletion zones in polyelectrolyte solutions in contact with like-charged flat surfaces are investigated. Using a coupled self-consistent field and Debye–Hückel approach an explicit expression for the thickness δ of the depletion layer is derived. It is found that δ ∼ δn + cκ^(-1), where δn is the depletion thickness at a neutral surface, c is a function of the electrostatic characteristics of the system and κ^(−1) the Debye length. It is argued that the theory still holds beyond the mean-field approximation, which is confirmed by quantitative agreement between our theoretical results and experiments.
... The effects are varied ranging from excluded volume, depletion effects to viscosity changes 10 , that in turn can affect cell physiology 11 . Crowding agent size and concentration have been previously reported to influence reaction rates in both experiment and theory as seen in case of the significant decrease in association rates of the β-Lactamase (TEM-1) and β-Lactamase inhibitor protein (BLIP) system with concentration in presence of small-sized crowdants but less so for large crowdants 12,13 . Amyloid fibre association rates in contrast were enhanced by small molecular weight crowding agents in a concentration dependent manner 14 . ...
Microtubule (MT) polymerization is regulated by biochemical as well as physical factors such as macromolecular crowding. Crowding agents or crowdants affect MT elongation rates differently depending on crowdant size due to opposing effects on polymerization: microviscosity reduces polymer elongation, while volume exclusion increases reaction rates by local concentration. In order to address how crowdant size and concentration collectively affect MT populations, we combine in vitro MT polymerization experiments with kinetic Monte Carlo simulations. Our experiments in bulk with nucleators validate decreasing MT elongation rates with increasing concentrations of small molecular weight crowdants in bulk assays and a corresponding increase for large crowdants. Kinetic Monte Carlo simulations can explain the result with packing fractions dependence of small as compared to large crowdants increasing microviscosity more dramatically. In contrast MT bulk polymerization rates in absence of nucleators increased with crowdant concentration, irrespective of their size, with a corresponding decrease in the critical concentration. Microscopy of filament growth dynamics demonstrates that small crowdants result in shorter filaments in a concentration dependent manner, consistent with their role in reducing elongation rates, but this decrease is compensated by increased number of filaments. Large crowdants increase the filament numbers while elongation is slightly decreased. Our results provide evidence for MT nucleation being rate-limited and elongation diffusion limited, resulting in differences in the effect of crowdant sizes on nucleation and elongation. These results are of general relevance to understand physical effects of crowding on collective cytoskeletal polymerization dynamics.
... The increase in concentration of GSH, PEG3, FIC70, and DEX70 means the protein and crowding molecules are eventually pushed inside each other's excluded volume regions via the osmotic pressure. The progressive decrease in I(0)/C and R g /R g (0) of D-Rec1 at higher concentrations of crowders, along with the increase in OD (aggregation), can be attributed to the excluded volume effects and solvent depletion around protein molecules (55,60). With the increase in concentration of a crowding agent, the solvent surrounding the D-Rec1 molecules gets depleted (because of the volume being taken up by crowding agents), which forces the protein molecules to decrease their R g by changing their conformation with more hydrophilic groups pushed toward the inside (core) and hydrophobic groups exposed outside (shell). ...
The consequences of crowding on the dynamic conformational ensembles of intrinsically disordered proteins (IDPs) remain unresolved because of their ultrafast motion. Here, we report crowder-induced interactions and conformational dynamics of a prototypical multistimuli-responsive IDP, Rec1-resilin. The effects of a range of crowders of varying sizes, forms, topologies, and concentrations were examined using spectroscopic, spectrofluorimetric, and contrast-matching small- and ultrasmall-angle neutron scattering investigation. To achieve sufficient neutron contrast against the crowders, deuterium-labeled Rec1-resilin was biosynthesized successfully. Moreover, the ab initio “shape reconstruction” approach was used to obtain three-dimensional models of the conformational assemblies. The IDP revealed crowder-specific systematic extension and compaction with the level of macromolecular crowding. Last, a robust extension-contraction model has been postulated to capture the fundamental phenomena governing the observed behavior of IDPs. The study provides insights and fresh perspectives for understanding the interactions and structural dynamics of IDPs in crowded states.
... Such an interpretation of the Stokes-Einstein relation successfully describes the behavior of proteins self-diffusion coefficient in semi-dilute and concentrated solutions [66][67][68]. ...
One of the commonly accepted approaches to estimate protein–protein interactions (PPI) in aqueous solutions is the analysis of their translational diffusion. The present review article observes a phenomenological approach to analyze PPI effects via concentration dependencies of self- and collective translational diffusion coefficient for several spheroidal proteins derived from the pulsed field gradient NMR (PFG NMR) and dynamic light scattering (DLS), respectively. These proteins are rigid globular α-chymotrypsin (ChTr) and human serum albumin (HSA), and partly disordered α-casein (α-CN) and β-lactoglobulin (β-Lg). The PPI analysis enabled us to reveal the dominance of intermolecular repulsion at low ionic strength of solution (0.003–0.01 M) for all studied proteins. The increase in the ionic strength to 0.1–1.0 M leads to the screening of protein charges, resulting in the decrease of the protein electrostatic potential. The increase of the van der Waals potential for ChTr and α-CN characterizes their propensity towards unstable weak attractive interactions. The decrease of van der Waals interactions for β-Lg is probably associated with the formation of stable oligomers by this protein. The PPI, estimated with the help of interaction potential and idealized spherical molecular geometry, are in good agreement with experimental data.
... The PEGs used are not solid shapes, but rather flexible, soft, and permeable cloud-like structures. PEGs are polyether and have been suggested to have a mesh-like structure above the semi-dilute regime (Kozer et al., 2007;Crowley et al., 2008;Lee et al., 2008). PEG fractions with molecular weights greater than a few thousand have long been known to have a large and mostly repulsive interaction with proteins and to induce macromolecular associations and compaction in qualitative agreement with crowding theory (Jarvis et al., 1990;Reddy et al., 1995). ...
Even though there are a great number of possible conformational states, how a protein generated as a linear unfolded polypeptide efficiently folds into its physiologically active form remained a fascinating and unanswered enigma inside crowded conditions of cells. In this study, various spectroscopic techniques have been exploited to know and understand the effect and mechanism of action of two different sizes of polyethylene glycols, or PEGs (molecular mass ∼10 and ∼20 kilo Daltons, kDa), on cytochrome c (cyt c). The outcomes showed that small size of the PEG leads to perturbation of the protein structure, and conversely, large size of the PEG has stabilizing effect on cyt c. Moreover, binding measurements showed that small size of PEG interacts strongly via soft interactions compared to the larger size of PEG, the latter being governed more by excluded volume effect or preferential exclusion from the protein. Overall, this finding suggests that conformations of protein may be influenced in cellular crowded conditions via interactions which depend upon the size of molecule in the environment. This study proposes that both volume exclusion and soft (chemical) interactions governs the protein’s conformation and functional activities. The cellular environment’s internal architecture as evident from crowder size and shape in this study has a significant role.
... As one striking example, the influence of polyethylene glycol on hydrophobic base-pair stacking in DNA was observed recently 23 . Besides that, effects of cellular confinement on kinetic interactions have been considered in different models dealing with fractal environments 24 and depletion interactions (representing an attractive force between particles that is based on the exclusion of solutes from the proximity of these two particles, also noted above) depending on the concentration and size of particles (inert molecules) constituting the solution under study 20,25 . In contrast to the complexity of the cellular environment, affinities of DNA-binding proteins are usually determined in vitro at experimental conditions comprising a dilute solution of a buffering agent and purified interaction partners stripped of their cellular surrounding. ...
The concept of Molecular Crowding depicts the high density of diverse molecules present in the cellular interior. Here, we determine the impact of low molecular weight and larger molecules on binding capacity of single-stranded DNA (ssDNA) to the cold shock protein B (CspB). Whereas structural features of ssDNA-bound CspB are fully conserved in crowded environments as probed by high-resolution NMR spectroscopy, intrinsic fluorescence quenching experiments reveal subtle changes in equilibrium affinity. Kinetic stopped-flow data showed that DNA-to-protein association is significantly retarded independent of choice of the molecule that is added to the solution, but dissociation depends in a nontrivial way on its size and chemical characteristics. Thus, for this DNA–protein interaction, excluded volume effect does not play the dominant role but instead observed effects are dictated by the chemical properties of the crowder. We propose that surrounding molecules are capable of specific modification of the protein’s hydration shell via soft interactions that, in turn, tune protein–ligand binding dynamics and affinity.
... Fig. 3 shows small decrease in stabilization in going from monomer to dimer to tetramer indicating that shape (oligomerization state) change of the crowder does not make significant changes in the free energy. We note in passing that the above trend for hard-sphere oligomers cannot be readily applied for polymeric crowders at high concentration (in the semi-dilute regime) as the overlapping fraction of polymers is often significant under these circumstances [65]. ...
Protein-protein association in vivo occur in a crowded and complex environment. Theoretical models based on hard-core repulsion predict stabilization of the product under crowded conditions. Soft interactions, on the contrary, can either stabilize or destabilize the product formation. Here we modeled protein association in presence of crowders of varying size, shape, interaction potential and used different mixing parameters for constituent crowders to study the influence on the association reaction. It was found that size is a more dominant factor in crowder-induced stabilization than the shape. Furthermore, in a mixture of crowders having different sizes but identical interaction potential, the change of free energy is additive of the free energy changes produced by individual crowders. However, the free energy change is not additive if two crowders of same size interact via different interaction potentials. These findings provide a systematic understanding of crowding influences in heterogeneous medium.
... Fig. 3 shows small decrease in stabilization in going from monomer to dimer to tetramer indicating that shape change of the crowder does not make significant changes in the free energy. We note in passing that the above trend for hard-sphere oligomers cannot be readily applied for polymeric crowders at high concentration (in the semi-dilute regime) as the overlapping fraction of polymers is often significant under these circumstances [58]. ...
Protein-protein association in vivo occur in a crowded and complex environment. Theoretical models based on hard-core repulsion predict stabilization of the product under crowded conditions. Soft interactions, on the contrary, can either stabilize or destabilize the product formation. Here we modeled protein association in presence of crowders of varying size, shape, interaction potential and used different mixing parameters for constituent crowders to study the influence on the association reaction. It was found that size a more dominant factor in crowder-induced stabilization than the shape. Furthermore, in a mixture of crowders having different sizes but identical interaction potential, the change of free energy is additive of the free energy changes produced by individual crowders. However, the free energy change is not additive if two crowders of same size interact via different interaction potentials. These findings provide a systematic understanding of crowding influences in heterogeneous medium.
... It is made available under a The copyright holder for this preprint this version posted August 18, 2020. . only Ash1 to compact, and the rest to expand, in line with other observations 27 . Larger polymers such as PEG2000 and Ficoll appear to compact the dimensions of all IDRs as shown for other disordered proteins 28 , with a sequence-dependent magnitude that is stronger for Ash1 and PUMA. ...
Intrinsically disordered protein-regions (IDRs) make up roughly 30% of the human proteome and are central to a wide range of biological processes. Given a lack of persistent tertiary structure, all residues in IDRs are, to some extent, solvent exposed. This extensive surface area, coupled with the absence of strong intramolecular contacts, makes IDRs inherently sensitive to their chemical environment. We report a combined experimental, computational, and analytical framework for high-throughput
characterization of IDR sensitivity. Our framework reveals that IDRs can expand or
compact in response to changes in their solution environment. Importantly, the direction and magnitude of conformational change depend on both protein sequence and cosolute identity. For example, some solutes such as short polyethylene glycol
chains exert an expanding effect on some IDRs and a compacting effect on others.
Despite this complex behavior, we can rationally interpret IDR responsiveness to solution composition changes using relatively simple polymer models. Our results imply that solution-responsive IDRs are ubiquitous and can provide an additional layer of regulation to biological systems.
... The synthetic compound is characterized by a polymer crossover concentration (f*) of 4% (w/w), which marks the transition from a semidilute to a crowded solvent regime. [31][32][33] As a consequence, all experiments were performed at 6% (w/w) PEG 4000 . ...
Mitochondrial transcript maturation in African trypanosomes requires RNA editing to convert nucleotide-deficient pre-mRNAs into translatable mRNAs. The different pre-mRNAs have been shown to adopt highly stable 2D-folds, however, it is not known whether these structures resemble the in vivo folds given the extreme “crowding” conditions within the mitochondrion. Here we analyze the effects of macromolecular crowding on the structure of the mitochondrial RPS12 pre-mRNA. We use polyethylene glycol as a macromolecular cosolute and monitor the structure of the RNA globally and with nucleotide resolution. We demonstrate that crowding has no impact on the 2D-fold and we conclude that the MFE-structure in dilute solvent conditions represents a good proxy for the folding of the pre-mRNA in its mitochondrial solvent context.
... At the majority of the concentrations of 8 kDa PEG used in this work, the solution is in a semidilute regime, exceeding the crossover concentration c * = N −4/5 ≈ 4% (w/v). It is at this concentration that the PEG molecules begin to overlap with one another, the beginnings of a mesh-like network where the molecules entangle one another (39). At these concentrations, crowding agent molecules may interact with pUC19 plasmids by inserting themselves into, or otherwise stabilizing, their unwinding regions, physically preventing the site from closing, and leaving them available for probe binding. ...
DNA unwinding is an important cellular process involved in DNA replication, transcription and repair. In cells, molecular crowding caused by the presence of organelles, proteins, and other molecules affects numerous internal cellular structures. Here, we visualize plasmid DNA unwinding and binding dynamics to an oligonucleotide probe as functions of ionic strength, crowding agent concentration, and crowding agent species using single-molecule CLiC microscopy. We demonstrate increased probe-plasmid interaction over time with increasing concentration of 8 kDa polyethylene glycol (PEG), a crowding agent. We show decreased probe-plasmid interactions as ionic strength is increased without crowding. However, when crowding is introduced via 10% 8 kDa PEG, interactions between plasmids and oligos are enhanced. This is beyond what is expected for normal in vitro conditions, and may be a critically important, but as of yet unknown, factor in DNA’s proper biological function in vivo. Our results show that crowding has a strong effect on the initial concentration of unwound plasmids. In the dilute conditions used in these experiments, crowding does not impact probe-plasmid interactions once the site is unwound.
... It is made available under a The copyright holder for this preprint this version posted August 18, 2020. . only Ash1 to compact, and the rest to expand, in line with other observations 27 . Larger polymers such as PEG2000 and Ficoll appear to compact the dimensions of all IDRs as shown for other disordered proteins 28 , with a sequence-dependent magnitude that is stronger for Ash1 and PUMA. ...
Intrinsically disordered proteins and protein-regions (IDRs) make up roughly 30% of the human proteome and play vital roles in a wide variety of biological processes. Given a lack of persistent tertiary structure, all of the residues in an IDR are, to some extent, solvent exposed. This extensive surface area, coupled with the absence of strong intramolecular contacts, makes IDRs inherently sensitive to their chemical environment. Despite this sensitivity, our understanding of how IDR structural ensembles are influenced by changes in their chemical environment is limited. This is particularly relevant given a growing body of evidence showing that IDR function is linked to the underlying structural ensemble. We develop and use a combined experimental, computational, and analytical framework for high-throughput characterization of IDR sensitivity we call solution space scanning. Our framework reveals that IDRs show sequence-dependent sensitivity to solution chemistry, with complex behavior that can be interpreted through relatively simple polymer models. Our results imply that solution-responsive IDRs are ubiquitous and can provide an additional layer of biological regulation.
... The influence of macromolecular crowding on the conformational properties of individual IDPs and on the binding interactions of folded proteins has been studied (32)(33)(34)(35), but little is known about the effect of crowding on the binding process involving IDPs, which is essential for many of their functions (9,36). Here, we aim to fill this gap with a systematic investigation of the effects of the size and concentration of polymeric crowding agents on the interaction between two IDPs by simultaneously monitoring complex stability, kinetics, and translational diffusion. ...
Significance
The molecular environment in a biological cell is much more crowded than the conditions commonly used in biochemical and biophysical experiments in vitro. It is therefore important to understand how the conformations and interactions of biological macromolecules are affected by such crowding. Addressing these questions quantitatively, however, has been challenging owing to a lack of sufficiently detailed experimental information and theoretical concepts suitable for describing crowding, especially when polymeric crowding agents and biomolecules are involved. Here, we use the combination of extensive single-molecule experiments with established and recent theoretical concepts to investigate the interaction between two intrinsically disordered proteins. We observe pronounced effects of crowding on their interactions and provide a quantitative framework for rationalizing these effects.
... Normally, polymer chains behave as coil-inducing attractive interactions between protein molecules at low-polymer concentration. However, at high polymer concentration, flexible coils elongate and enter space between the proteins, so that repulsive interaction are induced 50,51 . This explanation has been also confirmed by molecular dynamic simulations at different polymer concentration by Cao et al. 52 . ...
The crystallization of Anti-CD20, a full-length monoclonal antibody, has been studied in the PEG400/Na2SO4/Water system near Liquid-Liquid Phase Separation (LLPS) conditions by both sitting-drop vapour diffusion and batch methods. In order to understand the Anti-CD20 crystallization propensity in the solvent system of different compositions, we investigated some measurable parameters, normally used to assess protein conformational and colloidal stability in solution, with the aim to understand the aggregation mechanism of this complex biomacromolecule. We propose that under crystallization conditions a minor population of specifically aggregated protein molecules are present. While this minor species hardly contributes to the measured average solution behaviour, it induces and promotes crystal formation. The existence of this minor species is the result of the LLPS occurring concomitantly under crystallization conditions.
... Every formation of an encounter complex results in increased apparent size and slower rotational tumbling [4] [5] [6], manifested as reduced rotational diffusion, D rot . The effect on rotational diffusion differs from the translational diffusion, D t , in that spatial confinement per se does not have a direct impact on D rot : the cavities between larger structures and complexes enable relatively free rotation of small proteins, while sterically hindering translational movement, resulting in anomalous diffusion [7]. Nonetheless, all-atom MD simulations indicate that, taken over the whole ensemble, the crowded cytosol retards D t and D rot to a similar extent, on timescales so short that anomalous nonlinear displacements are not yet detectable [8]. ...
Random encounters between proteins in crowded cells are by no means passive, but found to be under selective control. This control enables proteome solubility, helps to optimise the diffusive search for interaction partners, to regulate the cell volume, and to allow for an adaptation to environmental extremes. Interestingly, the residues that modulate the random encounters act mesoscopically through protein net charge and exposed hydrophobicity, meaning that their impact and detailed signatures vary across organisms with different intracellular constraints. To examine such variations, we compare the diffusive behaviour of one bacterial and two human proteins in the E. coli and the human cytosols, using in-cell NMR relaxation. We find that proteins that ‘stick’ and whose signals become broadened beyond detection in E. coli are generally less restricted in mammalian cells. Also, the rotational correlation times in the mammalian cytosol are less sensitive to surface-charge mutations. This shows that, in terms of maintaining protein motions, the mammalian cytosol is more forgiving to surface alterations than E. coli cells. The cellular differences seem not linked to the proteome properties per se, but rather to a 6-fold difference in physiological protein concentrations. Taken together, the results outline a scenario in which the tolerant cytosol of mammalian cells, found in long-lived multicellular organisms, provides an enlarged evolutionary playground, where random protein-surface mutations are less deleterious than in short-generational bacteria.
... Concerning the E/DMSNs, all of them exhibited the mean diameter slightly higher than the corresponding DMSNs, thus implying that the guest molecules partially depositing on the surface of the DMSNs increased their aggregation. On the other hand, erianin depositing on the surface of the DMSNs might change the diffusion coefficient leading to increase the hydrodynamic radius [25,26]. This could be another reason for the increased mean diameter. ...
Background:
Psoriasis is a malignant skin disease characterized as keratinocyte hyperproliferation and aberrant differentiation. Our previous work reported that a bibenzyl compound, erianin, has a potent inhibitory effect on keratinocyte proliferation. To improve its poor water-solubility, increase anti- proliferation activity, and enhance the skin delivery, erianin loaded dendritic mesoporous silica nanospheres (E/DMSNs) were employed.
Results:
In this work, DMSNs with pore size of 3.5 nm (DMSN1) and 4.6 nm (DMSN2) were fabricated and E/DMSNs showed pore-size-dependent, significantly stronger anti-proliferative and pro-apoptotic effect than free erianin on human immortalized keratinocyte (HaCaT) cells, resulting from higher cellular uptake efficiency. In addition, compared to free erianin, treatment with E/DMSNs was more effective in reducing mitochondrial membrane potential and increasing cytoplasmic calcium levels, which were accompanied by regulation of mitochondria and endoplasmic reticulum stress (ERS) pathway. Porcine skin was utilized in the ex vivo accumulation and permeation studies, and the results indicated higher drug retention and less drug penetration in the skin when administered as the E/DMSNs-loaded hydrogel compared to the erianin-loaded hydrogel. Conlusions This work not only illustrated the further mechanisms of erianin in anti-proliferation of HaCaT cells but also offer a strategy to enhance the efficiency of erianin and the capacity of skin delivery through the DMSNs drug delivery systems.
... On the contrary, PEG200 molecules seemed to stabilize the aggregates, which occurred due to the FT stress. The reason for this might be based on the overall higher polymer concentrations in solution due to freeze concentration, resulting in a displacement of the PEG molecules from between the protein molecules instead of steric stabilization [71]. Hence, the restructuring of the PEG molecules seems to be not completely reversible. ...
Formulation conditions have a significant influence on the degree of freeze/thaw (FT) stress-induced protein instabilities. Adding cryoprotectants might stabilize the induced FT stress instabilities. However, a simple preservation of protein stability might be insufficient and further methods are necessary. This study aims to evaluate the addition of a heat cycle following FT application as a function of different cryoprotectants with lysozyme as exemplary protein. Sucrose and glycerol were shown to be the most effective cryoprotectants when compared to PEG200 and Tween20. In terms of heat-induced reversibility of aggregates, glycerol showed the best performance followed by sucrose, NaCl and Tween20 systems. The analysis was performed using a novel approach to visualize complex interplays by a clustering and data reduction scheme. In addition, solubility and structural integrity were measured and confirmed the obtained results.
Graphic abstract
It is now generally accepted that macromolecules do not act in isolation but “live” in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.
This series provides information on the nature of dyes, their harmful effects, and dye degrading techniques. The first volume of this series presents a fundamental concept of dye degradation. The information on target-oriented dye mitigation is intended to give readers a better understanding of the dye degradation process to sustain a healthy environment. Chapters present referenced information and highlight novel breakthroughs in the industry.
Key topics:
Foundations of Dye Knowledge: Evaluating Toxicity Nanotechnology Electrochemistry Catalytic Materials and Photocatalysis Microbial Biodegradation
This book serves as a foundational resource for researchers and students in chemistry and chemical engineering courses. It also serves as a reference for industry professionals who work with chemical dyes (for example in textile and plastic industries) and are engaged in the critical field of environmental remediation.
Complex coacervation refers to the liquid–liquid phase separation (LLPS) process occurring between charged macromolecules. The study of complex coacervation is of great interest due to its implications in the formation of membraneless organelles (MLOs) in living cells. However, the impacts of the crowded intracellular environment on the behavior and interactions of biomolecules involved in MLO formation are not fully understood. To address this knowledge gap, we investigated the effects of crowding on a model protein–polymer complex coacervate system. Specifically, we examined the influence of sucrose as a molecular crowder and polyethylene glycol (PEG) as a macromolecular crowder. Our results reveal that the presence of crowders led to the formation of larger coacervate droplets that remained stable over a 25-day period. While sucrose had a minimal effect on the physical properties of the coacervates, PEG led to the formation of coacervates with distinct characteristics, including higher density, increased protein and polymer content, and a more compact internal structure. These differences in coacervate properties can be attributed to the effects of crowders on individual macromolecules, such as the conformation of model polymers, and nonspecific interactions among model protein molecules. Moreover, our results show that sucrose and PEG have different partition behaviors: sucrose was present in both the coacervate and dilute phases, while PEG was observed to be excluded from the coacervate phase. Collectively, our findings provide insights into the understanding of crowding effects on complex coacervation, shedding light on the formation and properties of coacervates in the context of MLOs.
Proteins are one of the dynamic macromolecules that play a significant role in many physiologically important processes to sustain life on the earth. Proteins need to be properly folded into their active conformation to perform their function. Alteration in the protein folding process may lead to the formation of misfolded conformers. Accumulation of these misfolded conformers can result in the formation of protein aggregates which are attributed to many human pathological conditions including neurodegeneration, cataract, neuromuscular disorders, and diabetes. Living cells naturally have heterogeneous crowding environments with different concentrations of various biomolecules. Macromolecular crowding condition has been found to alter the protein conformation. Here in this review, we tried to show the relation between macromolecular crowding, protein aggregation, and its consequences.
Stimuli-responsive materials are exploited in biological, materials, and sensing applications. We introduce a new endogenous stimulus, biomacromolecule crowding, which we achieve by leveraging changes in thermoresponsive properties of polymers upon high concentrations of crowding agents. We prepare poly(2-oxazoline) amphiphiles that exhibit lower critical solution temperatures (LCST) in serum above physiological temperature. These amphiphiles stabilize oil-in-water nanoemulsions at temperatures below the LCST but are ineffective surfactants above the LCST, resulting in emulsion fusion. We find that the transformations observed upon heating nanoemulsions above their surfactant's LCST can instead be induced at physiological temperatures through the addition of polymers and protein, rendering thermoresponsive materials "crowding responsive." We demonstrate that the cytosol is a stimulus for nanoemulsions, with droplet fusion occurring upon injection into cells of living zebrafish embryos. This report sets the stage for classes of thermoresponsive materials to respond to macromolecule concentration rather than temperature changes.
The environmental condition is a critical regulation factor for protein behavior in solution. Several studies have shown that macromolecular crowders can modulate protein structures, interactions, and functions. Recent publications described the regulation of specific interaction by macromolecular crowders. However, the other category of protein-protein interaction, namely, the transient interaction, is rarely investigated, especially from the perspective of protein structure to study transient interactions between proteins. Here, we used nuclear magnetic resonance and small-angle X-ray/neutron scattering methods to structurally investigate the ensemble of the protein complex in dilute buffer and crowded environments. Histidine phosphocarrier protein (HPr) and the N-terminal domain of enzyme I (EIN) are the important components of the bacterial phosphotransfer system. Our results show that the addition of Ficoll-70 promotes HPr molecules to form the encounter complex with EIN maintained by long-range electrostatic interaction. However, when macromolecular crowder BSA is used, the soft interaction between BSA and HPr perturbs the active site of HPr, driving HPr to form an encounter complex with EIN at the weakly charged interface. Our results indicate that different macromolecular crowders could influence transient EIN-HPr interaction through different mechanisms and provide new insights into protein-protein interaction regulation in native environments.
The cellular environment is crowded by macromolecules of various sizes, shapes, and charges, which modulate protein structure, function and dynamics. Herein, we contemplated the effect of three different macromolecular crowders: dextran-40, ficoll-70 and PEG-35 on the structure, active-site conformational dynamics, function and relative domain movement of multi-domain human serum albumin (HSA). All the crowders used in this study have zero charges and similar sizes (at least in the dilute region) but different shapes and compositions. Some observations follow the traditional crowding theory. For example, all the crowders increased the α-helicity of HSA and hindered the conformational fluctuation dynamics. However, some observations are not in line with the expectations, as an increase in the size of HSA with PEG-35 and uncorrelated domain movement of HSA with ficoll-70 and PEG-35. The relative domain movement is correlated with the activity, suggesting that such moves are essential for protein function. The interaction between HSA and ficoll-70 is proposed to be hydrophobic in nature. Overall, our results provide a somewhat systematic study of the shape-dependent macromolecular crowding effect on various protein properties and present a possible new insight into the mechanism of macromolecular crowding.
Macromolecular crowding plays a crucial role in determining the dynamics in a living cell. We adopt Langevin dynamics simulations to investigate the anomalous diffusion dynamics of passive and active particles in a solution of polymer chains with tunable stiffness. The solution's anisotropic feature is modulated by changing both the polymer stiffness and volume fraction, where isotropic-to-nematic phase transition is involved. Our results demonstrate the significant impact of polymer flexibility on the dynamics of both passive and active probes. The distinct diffusion mechanism for an active particle is clarified by the interplay between polymer stiffness, crowdedness and activity. Polymer stiffness leads to a global inhibition effect on passive particle diffusion. The diffusion coefficient exhibits an intriguing non-monotonic variation at increasing polymer stiffness, which is due to the fact that the alignment of polymer chains is beneficial for diffusion along the nematic direction but unfavorable for that in the direction perpendicular to it. In sharp contrast, polymer stiffness plays a dominant role in facilitating active particle diffusion. Self-propulsion of the particle can utilize stiffness-induced elastic interactions more efficiently, which promotes its mobility in both directions. Meanwhile, an active particle might have a stronger ability to take advantage of the polymer alignment, contributing substantially enhanced diffusivity. In addition, the diffusion coefficient of an active particle is subject to a tendency of degeneration against varying volume fraction. This counter-intuitive behavior is due to the contrasting factors that increasing crowdedness induces a lower particle speed but a longer persistent motion time.
We derive a simple, yet accurate approximate mean-field expression for the depletion thickness δsf of a solution of dilute semi-flexible polymers next to a hard surface. In the case of a hard wall this equation has the simple form δsf = δ0[1 - tanh(psf/δ0)], where psf accounts for the degree of flexibility and δ0 is the depletion thickness in the case of fully flexible polymers. For fixed polymer coil size, increasing the chain stiffness leads to a decrease in the depletion thickness. The approach is also extended to include higher polymer concentrations in the semidilute regime. The analytical expressions are in quantitative agreement with numerical self-consistent field computations. A remarkable finding is that there is a maximum in the depletion thickness as a function of the chain stiffness in the semidilute concentration regime. This also means that depletion attractions between colloidal particles reach a maximum for a certain chain stiffness, which may have important implications for the phase stability of colloid-polymer mixtures. The derived equations could be useful for the description of interactions in- and phase stability of mixtures of colloids and semi-flexible polymers.
In biological systems, the synthesis of nucleic acids, such as DNA and RNA, is catalyzed by enzymes in various aqueous solutions. However, substrate specificity is derived from the chemical properties of the residues, which implies that perturbations of the solution environment may cause changes in the fidelity of the reaction. Here, we investigated non-promoter-based synthesis of RNA using T7 RNA polymerase (T7 RNAP) directed by an RNA template in the presence of polyethylene glycol (PEG) of various molecular weights, which can affect polymerization fidelity by altering the solution properties. We found that the mismatch extensions of RNA propagated downstream polymerization. Furthermore, PEG promoted the polymerization of non-complementary ribonucleoside triphosphates, mainly due to the decrease in the dielectric constant of the solution. These results indicate that the mismatch extension of RNA-dependent RNA polymerization by T7 RNAP is driven by the stacking interaction of bases of the primer end and the incorporated nucleotide triphosphates (NTP) rather than base pairing between them. Thus, proteinaceous RNA polymerase may display different substrate specificity with changes in dielectricity caused by molecular crowding conditions, which can result in increased genetic diversity without proteinaceous modification.
Nano-micron size fiber formation of UHMWPE (ultra-high molecular weight polyethylene) can be carried out by a high temperature (130 °C) electrospinning method. In this work, the effect of solution parameters on fiber formation by the electrospinning method is discussed. Two different grades of UHMWPE having a molecular weight (Mw) of 2.21 million g/mole (UM2.21) and 4.43 million g/mole (UM4.43) were chosen for the analysis. The solution properties of UHMWPE were investigated via rheology, which allowed the identification of the entanglement concentration before conducting the electrospinning process. The entanglement concentration (ce) for UM2.21 was 1.5 wt.%, while that for UM4.43 was 0.8 wt.%, respectively. The concentration exponent in semi dilute unentangled, and semi dilute entangled regimes was 1.23 and 2.59 for UM2.21 and 0.45 and 2.57 for UM4.43, respectively, in-line with de-Gennes’s scaling theory. SEM of the electrospun product revealed that defect-free smooth fiber morphology could only be obtained when the concentration of the solution is around 1.5–2.5 times the entanglement concentration.
Protein stability and performance in various natural and artificial systems incorporating many other macromolecules for therapeutic, diagnostic, sensor, and biotechnological applications attract increasing interest with the expansion of these technologies. Here we address the catalytic activity of lysozyme protein (LYZ) in the presence of a polyethylene glycol (PEG) crowder in a broad range of concentrations and temperatures in aqueous solutions of two different molecular mass PEG samples (Mw = 3350 and 10000 g/mol). The phase behavior of PEG-protein solutions is examined by using dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), while the enzyme denaturing is monitored by using an activity assay (AS) and circular dichroism (CD) spectroscopy. Molecular dynamic (MD) simulations are used to illustrate the effect of PEG concentration on protein stability at high temperatures. The results demonstrate that LYZ residual activity after 1 h incubation at 80 °C is improved from 15% up to 55% with the addition of PEG. The improvement is attributed to two underlying mechanisms. (i) Primarily, the stabilizing effect is due to the suppression of the enzyme aggregation because of the stronger PEG-protein interactions caused by the increased hydrophobicity of PEG and lysozyme at elevated temperatures. (ii) The MD simulations showed that the addition of PEG to some degree stabilizes the secondary structures of the enzyme by delaying unfolding at elevated temperatures. The more pronounced effect is observed with an increase in PEG concentration. This trend is consistent with CD and AS experimental results, where the thermal stability is strengthened with increasing of PEG concentration and molecular mass. The results show that the highest stabilizing effect is approached at the critical overlap concentration of PEG.
While extensive studies have been carried out to determine protein-RNA binding affinities, mechanisms, and dynamics in vitro, such studies do not take into consideration the effect of the many weak nonspecific interactions in a cell filled with potential binding partners. Here we experimentally tested the role of the cellular environment on affinity and binding dynamics between a protein and RNA in living U-2 OS cells. Our model system is the spliceosomal protein U1A and its binding partner SL2 of the U1 snRNA. The binding equilibrium was perturbed by a laser-induced temperature jump and monitored by Förster resonance energy transfer. The apparent binding affinity in live cells was reduced by up to 2 orders of magnitude compared to in vitro. The measured in-cell dissociation rate coefficients were up to 2 orders of magnitude larger, whereas no change in the measured association rate coefficient was observed. The latter is not what would be anticipated due to macromolecular crowding or nonspecific sticking of the uncomplexed U1A and SL2 in the cell. A quantitative model fits our experimental results, with the major cellular effect being that U1A and SL2 sticking to cellular components are capable of binding, just not as strongly as the free complex. This observation suggests that high binding affinities measured or designed in vitro are necessary for proper binding in vivo, where competition with many nonspecific interactions exists, especially for strongly interacting species with high charge or large hydrophobic surface areas.
A common feature of intrinsically disordered proteins (IDPs) is a disorder-to-order transition upon binding to other proteins, which has been tied to multiple benefits, including accelerated association rates or binding with low affinity, yet high specificity. Given the balanced equilibrium concentrations of folded and unfolded state of an IDP we asked the question if changes in the chemical environment, such as the presence of osmolytes or crowding agents, have a strong influence on the interaction of an IDP. Here, we demonstrate the impact of cosolutes on the interaction of the intrinsically disordered transcription factor c-Myb and its binding partner, the kinase-inducible interaction domain (KIX) of the CREB-binding protein. Temperature jump relaxation kinetics and microscale thermophoresis were employed in order to quantify the rate constants and the binding affinity of the c-Myb/KIX complex, respectively, in the presence of various cosolutes. We find the binding free energy of the c-Myb/KIX complex only marginally modulated by cosolutes, whereas the enthalpy and entropy of the interaction are very sensitive to the respective solvent conditions. For different cosolutes we observe substantial changes in enthalpy, both favorable and unfavorable, which are going with entropy changes largely compensating the enthalpy effects in each case. These characteristics might reflect a potential mechanism by which c-Myb offsets changes in the physico-chemical environment to maintain a roughly unaltered binding affinity.
Polymer translocation in complex environments is crucially important to many biological processes in life. In the present work, we adopted two-dimensional Langevin dynamics simulation to study the forced and unbiased polymer translocation dynamics in active and crowded media. Translocation time and probability are analyzed in terms of active force Fa, volume fraction f and also crowder size. The non-trivial active crowder size effect, activity-coupling effect as well as the novel mechanism of unbiased translocation between two active environments with different active particle sizes are clarified. Firstly, for forced translocation, we reveal an intriguing non-monotonic dependence of translocation time on crowder size in case of large activity. In particular, crowders in intermediate size similar as polymer segment are proven to be most favorable for translocation. Moreover, a facilitation-inhibition crossover of translocation time with increasing volume fraction is observed, indicating a crucial activity-crowding coupling effect. Secondly, for the unbiased translocation driven by different active crowder sizes, translocation probability demonstrates a novel turnover phenomenon, implying the appearance of an opposite directional preference as active force exceeds a critical value. Translocation time in both directions decreases monotonically with active force. The asymmetric activity effect together with entropic driving scenario provides a reasonable picture for the peculiar behavior observed in unbiased translocation.
Biology is beginning to appreciate the effects of the crowded and complex intracellular environment on the equilibrium thermodynamics and kinetics of protein folding. The next logical step involves the interactions between proteins. We review quantitative, wet-experiment based efforts aimed at understanding how and why high concentrations of small molecules, synthetic polymers, biologically relevant cosolutes and the interior of living cells affect the energetics of protein-protein interactions. We then address popular theories used to explain the effects and suggest expeditious paths for a more methodical integration of experiment and simulation.
Macromolecular crowding and the presence of surfaces can significantly impact the spatial organization of biopolymers. While the importance of crowding-induced depletion interactions in biology has been recognized, much remains to be understood about the effect of crowding on biopolymers such as DNA plasmids. A fundamental problem highlighted by recent experiments is to characterize the impact of crowding on polymer–polymer and polymer–surface interactions. Motivated by the need for quantitative insight, we studied flexible ring polymers in crowded environments using Langevin dynamics simulations. The simulations demonstrated that crowding can lead to compaction of isolated ring polymers and enhanced interactions between two otherwise repulsive polymers. Using umbrella sampling, we determined the potential of mean force (PMF) between two ring polymers as a function of their separation distance at different volume fractions of crowding particles, ϕ. An effective attraction emerged at ϕ ≈ 0.4, which is similar to the degree of crowding in cells. Analogous simulations showed that crowding can lead to strong adsorption of a ring polymer to a wall, with an effective attraction to the wall emerging at a smaller volume fraction of crowders (ϕ ≈ 0.2). Our results reveal the magnitude of depletion interactions in a biologically-inspired model and highlight how crowding can be used to tune interactions in both cellular and cell-free systems.
The polymerisation of nucleic acids is essential for copying genetic information correctly to the next generations, whereas mispolymerisation could promote genetic diversity. It is possible that in the prebiotic era, polymerases might have used mispolymerisation to accelerate the diversification of genetic information. Even in the current era, polymerases of RNA viruses frequently cause mutations. In this study, primer extension under different molecular crowding conditions was measured using T7 RNA polymerase as a model for the reaction in the prebiotic world. Interestingly, molecular crowding using 20 wt% poly(ethylene glycol) 2000 preferentially promoted the primer extensions with ATP and GTP by T7 RNA polymerase, regardless of Watson–Crick base-pairing rules. This indicates that molecular crowding decreases the dielectric constants in solution, resulting in enhancement of stacking interactions between the primer and an incorporated nucleotide. These findings suggest that molecular crowding could accelerate genetic diversity in the prebiotic world and may promote transcription error of RNA viruses in the current era.
Nearly all biological processes, including strictly regulated protein-protein interactions fundamental in cell signaling, occur inside living cells where the concentration of macromolecules can exceed 300 g/L. One such interaction is between a 7 kDa SH3 domain and a 25 kDa intrinsically disordered region of Son of Sevenless (SOS). Despite its key role in the mitogen activated protein kinase signaling pathway of all eukaryotes, most biophysical characterizations of this complex are performed in dilute buffered solutions where cosolute concentrations rarely exceed 10 g/L. Here, we investigate the effects of proteins, sugars, and urea, at high g/L concentrations, on the kinetics and equilibrium thermodynamics of binding between SH3 and two SOS-derived peptides using 19F NMR lineshape analysis. We also analyze the temperature-dependence, which enables quantification of the enthalpic and entropic contributions. The energetics of SH3-peptide binding in proteins differs from those in the small molecules we used as control cosolutes, demonstrating the importance of using proteins as physiologically-relevant cosolutes. Although the majority of the protein cosolutes destabilize the SH3-peptide complexes, the effects are non-generalizable and there are subtle differences, which are likely from weak nonspecific interactions between the test proteins and the protein crowders. We also quantify the effects of cosolutes on SH3 translational and rotational diffusion to rationalize the effects on association rate constants. The absence of a correlation between the SH3 diffusion data and the kinetic data in certain cosolutes suggests that the properties of the peptide in crowded conditions must be considered when interpreting energetic effects. These studies have implications for understanding protein-protein interactions in cells and show the importance of using physiologically-relevant cosolutes for investigating macromolecular crowding effects.
Intrinsically disordered proteins (IDPs) comprise ~30–40% of the proteome, have key roles in cellular processes, and have been reported to be involved in stress regulation working in synergy with osmolytes. Osmolytes are known to accumulate against various stresses in living systems and are known to stabilize the native conformation of globular proteins. However, little is known of their effect on IDPs and their mechanism of action is unclear. We have investigated the effect of a series of polyol osmolytes on the conformation, aggregation and fibrillation properties of the IDPs α and β-synuclein, involved in Parkinson's disease, using fluorescence, CD, light scattering and TEM. We observe inhibition of fibril and aggregate formation with increasing concentration as well as the number of hydroxyl groups in polyols as observed by light scattering measurements which correlates well with the increase in viscosity of solution with increasing number of OH groups in them. However, ThT assay, while indicating suppression of fibril formation at various concentrations of polyols, shows enhanced fibrillation at some other concentrations which could be due to the heterogeneity of the species formed that are ThT insensitive. Fibril formation was, thus, probed by using Nile red fluorescence which showed sensitivity towards the species formed. ANS binding fluorescence also indicates a decrease in the hydrophobicity of the fibrils with increasing number of OH groups in polyols. Polyols do not have any effect on the fibrillation of β-syn but lead to enhanced amorphous aggregate formation in presence of Ethylene Glycol and Glycerol and a reduction in the presence of Sorbitol. The net free energy of transfer of the proteins from water to Sorbitol is large and positive while it is relatively negligible in the case of Glycerol suggestive of greater preferential exclusion effect of Sorbitol in comparison with Glycerol in the case of IDPs as well. The results overall show differential and complex effect of osmolytes towards the fibrillation/aggregation properties of the two IDPs and suggest that an appropriate balance between the concentration and type of polyol or osmolyte would be required for the survival of organisms rich in IDPs under various stress conditions.
It is now well appreciated that the crowded intracellular environment significantly modulates an array of physiological processes including protein folding-unfolding, aggregation, and dynamics to name a few. In this work we have studied the dynamics of domain I of the protein human serum albumin (HSA) in its urea-induced denatured states, in the presence of a series of commonly used macromolecular crowding agents. HSA was labeled at Cys-34 (a free cysteine) in domain I with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) to act as a solvation probe. In partially denatured states (2-6 M urea), lower crowder concentrations (~ < 125 g/L) induced faster dynamics, while the dynamics became slower beyond 150 g/L of crowders. We propose that this apparent switch in dynamics is an evidence of a crossover from soft (enthalpic) to hard-core (entropic) interactions between the protein and crowder molecules. That soft interactions are also important for the crowders used here was further confirmed by the appreciable shift in the wavelength of the emission maximum of BADAN, in particular for PEG8000 and Ficoll 70 at concentrations where the excluded volume effect is not dominant.
Although it is well known that the environment of mitochondria is a densely packed network of macromolecules, the kinetics of the essential metabolic enzyme, citrate synthase, has only been studied under dilute conditions. To understand how this crowded environment impacts the behavior of citrate synthase, Michaelis-Menten kinetics were measured spectrophotometrically in the presence of synthetic crowders as a function of size, concentration, and identity. The biggest factor contributing to crowding effects was the overlap concentration (c*), the concentration above which polymers begin to interact. The presence of the crowder, dextran, decreased the maximum rate of the reaction by about 20% in the dilute regime (< c*) and 40% in the semi-dilute regime (>c*) regardless of polymer size. The disparate effects observed from different crowding agents of similar size also reveal the importance of transient interactions from crowding.
Many human DNA repair proteins have disordered domains at their N- or C-termini with poorly defined biological functions. We recently reported that the partially structured N-terminal domain (NTD) of human uracil DNA glycosylase 2 (hUNG2), functions to enhance DNA translocation in crowded environments and also targets the enzyme to single-stranded/double-stranded DNA junctions. To understand the structural basis for these effects we now report high-resolution heteronuclear NMR studies of the isolated NTD in the presence and absence of an inert macromolecular crowding agent (PEG8K). Compared to dilute buffer, we find that crowding reduces the degrees of freedom for the structural ensemble, increases the order of a PCNA binding motif and dramatically promotes binding of the NTD for DNA through a conformational selection mechanism. These findings shed new light on the function of this disordered domain in the context of the crowded nuclear environment.
The effects of 17 kinds of additive mixtures have been studied on refolding and aggregation of a model protein, lysozyme. Most of the prepared mixtures were efficient in inhibiting aggregation of the protein and surprisingly, 4 novel additive mixtures, i.e. lactic acid: L‐arginine, lactic acid: L‐glutamine, choline chloride: lactic acid and imidazolium salt: β‐cyclodextrin as well as choline chloride: urea exhibited a more remarkable efficacy in suppressing aggregation. Among these, lactic acid: L‐arginine was identified as the most efficient additive, and lactic acid: L‐glutamine and choline chloride: lactic acid were inefficient to recover the enzyme activity. In contrast, choline chloride: ethylene glycol: imidazole, choline chloride: glycerol: imidazole, imidazole: betaine: ethylene glycol were found to be less effective mixtures in preventing enzyme aggregation. Totally, it was demonstrated that the protective effects of the mixtures were improved as their concentrations increased. The improvement was more remarkable for imidazolium salt: β‐cyclodextrin and choline chloride: urea, where the denatured lysozyme was reactivated and recovered up to 85% of its initial activity by enhancing their concentrations from 1 to 5% (V/V). It is suggested that such solution additives may be further employed as artificial chaperones in order to assist protein folding and stability. This article is protected by copyright. All rights reserved
The thermodynamic activity of proteins in solution is substantially altered by the addition of unreactive or 'inert' macromolecules occupying more than a few percent of total solution volume. Approximate theoretical models of this effect have been formulated using a simplified geometrical representation of molecular shapes. These models predict that under certain conditions, the structure and function of proteins in physiological media with a high total macromolecular content may be qualitatively different than in dilute solution. Experimental studies of the effect of 'inert' macromolecules on protein structure and/or function are reviewed, and it is found that under favorable circumstances the simplified models can provide a satisfactory semiquantitative description of the data.
There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the number of ways it is possible to change the temperature, pressure, and chemical potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.
A protein design strategy was developed to specifically enhance the rate of association (k(on)) between a pair of proteins without affecting the rate of dissociation (k(off)). The method is based on increasing the electrostatic attraction between the proteins by incorporating charged residues in the vicinity of the binding interface. The contribution of mutations towards the rate of association was calculated using a newly developed computer algorithm, which predicted accurately the rate of association of mutant protein complexes relative to the wild type. Using this design strategy, the rate of association and the affinity between TEM1 beta-lactamase and its protein inhibitor BLIP was enhanced 250-fold, while the dissociation rate constant was unchanged. The results emphasize that long range electrostatic forces specifically alter k(on), but do not effect k(off). The design strategy presented here is applicable for increasing rates of association and affinities of protein complexes in general.
Phase diagrams of biological macromolecules are governed by an appropriate combination of interaction potentials in solution. Repulsive regimes favor solubility, whereas the presence of attractive potentials may induce a variety of phase transitions, including the desired macromolecular crystallization. The forces at work may be analyzed with a combination of small angle X-ray scattering and of numerical treatments. From the results obtained with a variety of model systems, the respective advantages and drawbacks of using monovalent salts or PEGs as crystallizing agents are discussed.
We present the experimental and theoretical background of a method to characterize the protein-protein attractive potential induced by one of the mostly used crystallizing agents in the protein-field, the poly(ethylene glycol) (PEG). This attractive interaction is commonly called, in colloid physics, the depletion interaction. Small-Angle X-ray Scattering experiments and numerical treatments based on liquid-state theories were performed on urate oxidase-PEG mixtures with two different PEGs (3350 Da and 8000 Da). A "two-component" approach was used in which the polymer-polymer, the protein-polymer and the protein-protein pair potentials were determined. The resulting effective protein-protein potential was characterized. This potential is the sum of the free-polymer protein-protein potential and of the PEG-induced depletion potential. The depletion potential was found to be hardly dependent upon the protein concentration but strongly function of the polymer size and concentration. Our results were also compared with two models, which give an analytic expression for the depletion potential.
We discuss structural correlations in mixtures of free polymer and
colloidal particles on the basis of a microscopic, two-component
liquid-state integral equation theory. Whereas in the case of polymers
much smaller than the spherical particles the relevant polymer degree of
freedom is the centre of mass, for polymers larger than the (nano-)
particles, conformational rearrangements need to be considered. They
have the important consequence that the polymer depletion layer exhibits
two widely different length scales, one of the order of the particle
radius, the other of the order of the polymer radius or the
polymer-density screening length in dilute or semidilute concentrations,
respectively. Because we find a spinodal instability (mostly) below the
overlap concentration, the latter length is (mostly) set by the radius
of gyration. As a consequence of the structure of the depletion layer,
the particle-particle correlations depend on both length scales for
large polymers. Because of the high local compressibility of large
polymers, the local depletion layer is a strong function of particle
density, but a weak function of polymer concentration. The amplitude of
the long-ranged tail of the depletion layer also depends asymptotically
only on the colloid concentration, while the range increases upon
approaching the (mean-field) spinodal. The colloid correlations may be
understood as characteristic for particles with a short-ranged potential
when small polymers are added, and as characteristic for particles with
a long-ranged, van der Waals-like attraction when the added free polymer
coils are much larger. Small polymers fill the voids between the
particles rather homogeneously, exhibiting correlations inside the mesh
(which gets squeezed by the colloids) and Porod-like correlations for
larger distances. The structure factor of large polymers, however,
exhibits no ramified mesh and becomes a Lorentzian characterized by the
mixture correlation length, which diverges at the spinodal.
A molecular dynamics simulation study of the influence of a polymer melt matrix consisting of bead-necklace polymers on the effective interaction between two spherical nanoparticles was performed. The potential of mean force (POMF) between the two nanoparticles as well as entropy and energy contributions to the POMF was determined as a function of nanoparticle separation. The role of energy on the POMF was investigated by varying the strength of the polymer-nanoparticle interaction and comparing structure and POMF with those obtained for an athermal model. All features of the POMF as a function of nanoparticle separation were found to be strongly correlated with the polymer matrix density, the structure of the polymer at the nanoparticle interface, and the structure of the polymer in the interparticle region. The POMF was not found to correlate with polymer chain dimensions (e.g., radius of gyration) in contrast to colloidal suspensions in dilute and semidilute solutions. Both energy and entropy effects were found to make important contributions to the POMF. For the athermal system, where all matrix-induced interactions are entropic in nature, the nanoparticle POMF was found to exhibit qualitatively different behavior from that of the energetic systems.
Monte Carlo simulations of the fluctuating bond lattice model are used to determine the force between colloidal particles immersed in a nonadsorbing polymeric fluid. Monodisperse systems with chain lengths of 20 to 100 segments are studied at occupation fractions ranging from 0.1 to 0.6, covering the semidilute and dense regimes. The variation of the force with concentration, particle diameter, and interparticle separation is in qualitative agreement with predictions of scaling theory and of integral equations for the colloid–polymer system. In semidilute solutions the force is purely attractive and displays an approximately linear dependence upon separation for small colloid separations. At higher concentrations the force is repulsive, for certain separations.
The interactions between globular proteins in the presence of poly (ethylene glycol) (PEG) are probed through the measurement of the protein solution second virial coefficient (B2). The solution properties of PEG are characterized for four molecular weights (400, 1000, 6000, and 12 000), providing an opportunity for quantitative comparison of measurements and theoretical predictions of B2. PEG displays a buffer and molecular weight-dependent lower critical solution temperature. As the polymer solution approaches phase separation, the consequences of depletion attractions increase significantly. For lysozyme and bovine serum albumin in sulfate buffers with PEG, B2 is not well described by standard depletion models. This failure is accentuated in acetate buffers where B2 is a nonmonotonic function of polymer concentration. The attractive minima in B2 are closely associated with the proximity of the heating-induced phase separation of aqueous PEG solutions. The experimental data for both proteins in the presence of PEG are well captured by the thermal polymer reference interaction site model for depletion interactions where the polymer density fluctuation correlation length is treated as a function of temperature, polymer concentration, and molecular weight. © 2000 American Institute of Physics.
The treatment of diffusion‐controlled bimolecular chemical reactions is extended to molecules with axially symmetrical distributions of reactivity on their spherical surfaces. Whereas an explicit solution exists for the time‐dependent effective rate constant at small times, the physically more interesting solution at large times can be obtained only approximately (though to any required degree of accuracy) by solving sets of linear equations.
We develop an analytic integral equation theory for treating polymer-induced effects on the structure and thermodynamics of dilute suspensions of hard spheres. Results are presented for the potential of mean force, free energy of insertion per particle into a polymer solution, and the second virial coefficient between spheres. The theory makes predictions for all size ratios between the spheres and the polymer coil dimension. Based on the Percus–Yevick (PY) closure, the attractive polymer-induced depletion interaction is predicted to be too weak under athermal conditions to induce a negative value for the second virial coefficient, B2cc, between spheres in the colloidal limit when the spheres are much larger than the coil size. A nonmonotonic dependence of the second virial coefficient on polymer concentration occurs for small enough particles, with the largest polymer-mediated attractions and most negative B2cc occurring near the dilute–semidilute crossover concentration. Predictions for the polymer-mediated force between spheres are compared to the results of computer simulations and scaling theory. © 1998 American Institute of Physics.
Scitation is the online home of leading journals and conference proceedings from AIP Publishing and AIP Member Societies
The forces between polymer-bearing surfaces in motion with respect to each other in a liquid medium are very different from their equilibrium interactions and control effects ranging from colloidal rheology to lubrication and wear. Several aspects of these forces are reviewed, both when surfaces are approaching or receding (lubrication forces) or when they slide past each other (shear and frictional forces). Lubrication forces are considered in different solvency conditions and for different types of polymer attachment to the surfaces, particularly in compressed layers, where viscous segmental drag on the flowing liquid dominates. Frictional forces between compressed, rubbing solid surfaces may be dramatically reduced by the attachment of solvated chain layers, which are able to sustain large normal loads while retaining a fluid interfacial layer. The coupling between sliding motion and normal forces is described, and the relation between the shear rates required to stretch chains and the chain relaxation rate is analyzed.
Dynamic light scattering has been used to study the diffusion of polystyrene (PS) latex spheres (≈0.2 μm radius) in poly(methyl methacrylate) (PMMA) solutions at 25 °C. The weight-average molecular weight of the PMMA was 350 000. The sign and magnitude of the deviations from the Stokes−Einstein (SE) equation varied dramatically with solvent quality. Positive deviations from SE behavior (ηDsphere/η0D0 > 1) were observed in good solvents for PMMA, such as dimethylformamide (DMF) and tetrahydrofuran (THF). We argue that these positive deviations are a result of a layer of solution locally surrounding the latex spheres that is rich in solvent and deficient in PMMA. This “depletion layer” is likely caused by a combination of entropic repulsion between the matrix PMMA and the latex spheres, and most importantly the immiscibility of PMMA and PS. A negative deviation (ηDsphere/η0D0 < 1) by almost of factor of 3 from the SE equation was observed in a dioxane−water mixed solvent, which is a ϑ-solvent for PMMA at 25 °C. PMMA adsorption onto the latex spheres is argued to occur under these unfavorable solvent conditions. At high matrix concentrations, entanglements of the adsorbed PMMA with free PMMA in solution likely occur. A very slow relaxation mode, in addition to the mode associated with sphere diffusion, was present in the CONTIN analyses of the PMMA/PS latex/dioxane−water system. It is possible that this mode is due to PMMA clusters, or less likely, bridged PS latex moieties.
Fluorescence correlation spectroscopy (FCS) is an experimental technique using statistical analysis of the fluctuations of fluorescence in a system in order to decipher dynamic molecular events, such as diffusion or conformational fluctuations of biomolecules. First introduced by Magde et al to measure the diffusion and binding of ethidium bromide onto double-stranded DNA, the technique has been undergoing a renaissance since 1993 with the implementation of confocal microscopy FCS. Since then, a flurry of experiments has implemented FCS to characterize the photochemistry of dyes, the translational and rotational mobilities of fluorescent molecules, as well as to monitor conformational fluctuations of green fluorescent proteins and DNA molecules.
In this review, we present the analytical formalism of an FCS measurement, as well as practical considerations for the design of an FCS setup and experiment. We then review the recent applications of FCS in analytical chemistry, biophysics and cell biology, specifically emphasizing the advantages and pitfalls of the technique compared to alternative spectroscopic tools. We also discuss recent extensions of FCS in single-molecule spectroscopy, offering alternative data processing of fluorescence signals to glean more information on the kinetic processes.
Association of a protein complex follows a two-step mechanism, with the first step being the formation of an encounter complex that evolves into the final complex. Here, we analyze recent experimental data of the association of TEM1-β-lactamase with BLIP using theoretical calculations and simulation. We show that the calculated Debye–Hückel energy of interaction for a pair of proteins during association resembles an energy funnel, with the final complex at the minima. All attraction is lost at inter-protein distances of 20 Å, or rotation angles of >60° from the orientation of the final complex. For faster-associating protein complexes, the energy funnel deepens and its volume increases. Mutations with the largest impact on association (hotspots for association) have the largest effect on the size and depth of the energy funnel. Analyzing existing evidence, we suggest that the transition state along the association pathway is the formation of the final complex from the encounter complex. Consequently, pairs of proteins forming an encounter complex will tend to dissociate more readily than to evolve into the final complex. Increasing directional diffusion by increasing favorable electrostatic attraction results in a faster forming and slower dissociating encounter complex. The possible applicability of electrostatic calculations for protein–protein docking is discussed. Proteins 2001;45:190–198. © 2001 Wiley-Liss, Inc.
The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and beta-lactoglobulin with polyethylene glycols (PEGs) of molecular weight 200-6,000 have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein. The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by Rg = aM0.55. The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24, 6756-6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.
A new approach to the calculation of bimolecular association constants for partially diffusion-limited reactions between asymmetric species (e.g. the ligand binding site of a macromolecule covers only a portion of its surface) is presented. The usual formulation, which is almost always analytically intractable, is based on the solution of a steady-state rotational-translational diffusion equation subject to the mixed boundary conditions that (A) the ligand concentration vanishes over the reactive part of the macromolecular surface and (B) the flux vanishes over the remainder. We show that if A is replaced by the requirement that the flux is a constant over the reactive part of the macromolecular surface and this constant is evaluated by requiring the concentration to vanish on the average over the sink region, a whole class of problems can be solved analytically. We consider both the translational and rotational diffusion of the reactants and treat partially diffusion-controlled reactions using the so-called radiation boundary condition. To establish the validity of our approach, we study a simple model using the usual mixed as well as our boundary conditions. As illustrations of our method, we analytically solve and analyze the properties of two models that have been previously studied using numerical methods.
A unified model is presented for protein-protein association processes that are under the influences of electrostatic interaction and diffusion (e.g., protein oligomerization, enzyme catalysis, electron and energy transfer). The proteins are modeled as spheres that bear point charges and undergo translational and rotational Brownian motion. Before association can occur the two spheres have to be aligned properly to form a reaction complex via diffusion. The reaction complex can either go on to form the product or it can dissociate into the separate reactants through diffusion. The electrostatic interaction, like diffusion, influences every step except the one that brings the reaction complex into the product. The interaction potential is obtained by extending the Kirkwood-Tanford protein model (Tanford, C., and J. G. Kirkwood. 1957. J. Am. Chem. Soc. 79:5333-5339) to two charge-embedded spheres and solving the consequent equations under a particular basis set. The time-dependent association rate coefficient is then obtained through Brownian dynamics simulations according an algorithm developed earlier (Zhou, H.-X. 1990. J. Phys. Chem. 94:8794-8800). This method is applied to a model system of the cytochrome c and cytochrome c peroxidase association process and the results confirm the experimental dependence of the association rate constant on the solution ionic strength. An important conclusion drawn from this study is that when the product is formed by very specific alignment of the reactants, as is often the case, the effect of the interaction potential is simply to scale the association rate constant by a Boltzmann factor. This explains why mutations in the interface of the reaction complex have strong influences on the association rate constant whereas those away from the interface have minimal effects. It comes about because the former mutations change the interaction potential of the reaction complex significantly and the latter ones do not.
The rates of folding of wild-type chymotrypsin inhibitor 2 (CI2) (t1/2 = 12 ms) and of faster (t1/2 = 2 ms) and slower (t1/2 = 350 ms) folding mutants are accelerated in parallel by increasing concentrations of sucrose, despite the increases in viscosity. At a viscosity 26 times that of water, the folding rate constant of wild-type CI2 is accelerated four-fold (t1/2 = 2.7 ms). From this, we can estimate that the diffusional chain collapse in CI2 occurs in less than 100 micros in water, and is not rate-determining in folding.
Biological macromolecules evolve and function within intracellular environments that are crowded with other macromolecules. Crowding results in surprisingly large quantitative effects on both the rates and the equilibria of interactions involving macromolecules, but such interactions are commonly studied outside the cell in uncrowded buffers. The addition of high concentrations of natural and synthetic macromolecules to such buffers enables crowding to be mimicked in vitro, and should be encouraged as a routine variable to study. The stimulation of protein aggregation by crowding might account for the existence of molecular chaperones that combat this effect. Positive results of crowding include enhancing the collapse of polypeptide chains into functional proteins, the assembly of oligomeric structures and the efficiency of action of some molecular chaperones and metabolic pathways.
The first sign that PEO is “unusual” can be sensed from it’s unexpected water solubility or “hydrophilicity.” Thus, poly(butylene oxide), –[CH2–CH2–CH2–O–]n–, having one more methylene (CH2) group, is hydrophobic and insoluble in water, as might be expected. But so is poly(methylene oxide), –[CH2–O–]n–, which has one less methylene group.
The structure of a protein-protein interaction, its affinity and thermodynamic characteristics depict a 'frozen' state of a complex. This picture ignores the kinetic nature of complex formation and dissociation, which are of major biological and biophysical interest. This review highlights recent advances in deciphering the kinetic pathway of protein-protein complexation, the nature of the encounter complex, transition state and intermediate along the reaction, and the effects of mutation, viscosity, pH and salt on association.
Physiological media constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo. Measuring protein-protein interaction in crowded solutions can mimic this environment. In this work we follow the process of protein-protein association and its rate constants (k(on)) of the beta-lactamase (TEM)-beta-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents. In all crowded solutions (0-40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute k(on), but not k(off) values, were found to be slower as compared to buffer. However, there is a fundamental difference between low and high mass crowding agents. In the presence of low mass crowding agents and Haemaccel k(on) depends inversely on the solution viscosity. In high mass polymer solutions k(on) changes only slightly, even at viscosities 12-fold higher than water. The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length (L) and its persistence length (Lp). Polymers that are long enough to form a flexible coil (L/Lp > 2) behave as high molecular mass polymers and those who are unable to do so (L/Lp < 2) behave as low molecular mass polymers. We concluded that although polymers solution are crowded, this property is not uniform; i.e. there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment. This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton. To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed k(on) of wt-TEM and three BLIP analogs with up to 100-fold increased values of k(on) due to electrostatic steering. Faster associating BLIP variants keep their relative advantage in all crowded solutions, including Haemaccel. This result suggests that faster associating protein complexes keep their advantage also in complex environment.
A number of different diseases are collectively described as the amyloid disorders because they result from the misfolding
and aggregation of proteins into amyloid fibrils or their precursors that are deposited in various tissues where they wreak
havoc. In his Perspective,
Dobson
provides an overview of research into amyloid diseases and discusses possible therapeutic interventions that could prevent
or reverse the aggregation process.
The structure, effective forces, and thermodynamics in dense polymer-particle mixtures were analyzed using the Polymer Reference Interaction Site Model (PRISM) theory. Depletion interactions, induced by polymer were quantified through the particle-particle pair correlation function and potential of mean force. It was found that a transition from monotonic decaying, attractive depletion interactions to much stronger repulsive-attractive oscillatory depletion forces took place at nearly the semidilute-concentrated solution boundary. The results show that the particle-particle potential of mean force shows multiple qualitatively different behaviours which rely on the strength and spatial range of the polymer-particle attraction.
It is now clear that a significant fraction of eukaryotic genomes encode proteins with substantial regions of disordered structure. In spite of the lack of structure, these proteins nevertheless are functional; many are involved in critical steps of the cell cycle and regulatory processes. In general, intrinsically disordered proteins interact with a target ligand (often DNA) and undergo a structural transition to a folded form when bound. Several features of intrinsically disordered proteins make them well suited to interacting with multiple targets and to cell regulation. New algorithms have been developed to identify disordered regions of proteins and have demonstrated their presence in cancer-associated proteins and proteins regulated by phosphorylation.
The reaction between cytochrome f and plastocyanin is a central feature of the photosynthetic electron-transport system of all oxygenic organisms. We have studied the reaction in solution to understand how the very weak binding between the two proteins from Phormidium laminosum can nevertheless lead to fast rates of electron transfer. In a previous publication [Schlarb-Ridley, B. G., et al. (2003) Biochemistry 42, 4057-4063], we suggested that the reaction is diffusion-controlled because of a strong effect of viscosity of the medium. The effects of viscosity and temperature have now been examined in detail. High molecular mass viscogens (Ficoll 70 and Dextran 70), which might mimic in vivo conditions, had little effect up to a relative viscosity of 4. Low molecular mass viscogens (ethane diol, glycerol, and sucrose) strongly decreased the bimolecular rate constant (k(2)) over a similar viscosity range. The effects correlated well with the viscosities of the solutions of the three reagents but not with their dielectric constants or molalities. A power law dependence of k(2) on viscosity suggested that k(2) depends on two viscosity-sensitive reactions in series, while the reverse reactions are little affected by viscosity. The results were incompatible with diffusion control of the overall reaction. Determination of the effect of temperature on k(2) gave an activation enthalpy, DeltaH(++) = 45 kJ mol(-)(1), which is also incompatible with diffusion control. The results were interpreted in terms of a model in which the stable form of the protein-protein complex requires further thermal activation to be competent for electron transfer.
The concept of excluded volume and possible effects of excluded volume on the reactivity of macromolecules in highly volume-occupied or "crowded" media are introduced and briefly summarized. Theoretical and experimental studies of the effect of crowding on protein folding and unfolding, and on the effect of crowding on protein association and aggregation, are reviewed. Possible effects of the effect of crowding on an initially native protein that can undergo unfolding, self-association of native protein, and/or aggregation of non-native protein are considered.
The association of two proteins is preceded by a mutual diffusional search in solution. The role of translational and rotational diffusion in this process has been studied theoretically for many years. However, systematic experimental verification of theoretical results is still lacking. We report here measurements of association rates of the proteins beta-lactamase (TEM) and beta-lactamase inhibitor protein (BLIP) in solutions of glycerol and poly(ethylene glycol) of increasing viscosity. We also measured translational and rotational diffusion in the same solutions, using fluorescence correlation spectroscopy and fluorescence anisotropy, respectively. It is found that in glycerol both translational and rotational diffusion rates are inversely dependent on viscosity, as predicted by the classical Stokes-Einstein relations, while the association rate depends nonlinearly on viscosity. In contrast, the association rate depends only weakly on the viscosity of the polymer solutions, which results in a similar weak dependence of k(on) on viscosity. The data are modeled using the theory of diffusion-limited association. Deviations from the theory are explained by a short-range solute-induced repulsion between the proteins in glycerol solution and an attractive depletion interaction generated by the polymers. These results open the way to the creation of a unified framework for all nonspecific effects involved in the protein association process, as well as to better theoretical understanding of these effects. Further, they reflect on the complex factors controlling protein association within the crowded environment of cells and suggest that a high concentration of macromolecules does not significantly impede protein association.
The Theory of Polymer Dynamics (The International Series of Monographs on Physics)
- M Doi
- S F Edwards
Doi, M., and S. F. Edwards. 1988. The Theory of Polymer Dynamics (The International Series of Monographs on Physics). Oxford Univer- sity Press, New York.
Protein chemistry. In the footsteps of alchemists
- Dobson