Crowder Concentration Research Articles

Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu.

The natural cellular milieu is crowded by large quantities of various biological macromolecules. This complex environment is characterized by a limited amount of unoccupied space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly packed cellular environment is poorly mimicked by traditional physiological conditions, where low concentrations of a protein of interest are analyzed in slightly salted aqueous solutions. An alternative is given by the use of a model crowded milieu, where a protein of interest is immersed in a solution containing high concentrations of various polymers that serve as model crowding agents. An expected outcome of the presence of such macromolecular crowding agents is their ability to increase conformational stability of a globular protein due to the excluded volume effects. In line with this hypothesis, the behavior of a query protein should be affected by the hydrodynamic size and concentration of an inert crowder (i.e., an agent that does not interact with the protein), whereas the chemical nature of a macromolecular crowder should not play a role in its ability to modulate conformational properties. In this study, the effects of different crowding agents (polyethylene glycols (PEGs) of various molecular masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70) on the spectral properties and unfolding–refolding processes of the super-folder green fluorescent protein (sfGFP) were investigated. sfGFP is differently affected by different crowders, suggesting that, in addition to the expected excluded volume effects, there are some changes in the solvent properties.

Protein Composition Determines the Effect of Crowding on the Properties of Disordered Proteins

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

Small Angle Neutron Scattering of the Intrinsically Disordered Protein FlgM under Crowded Conditions

Using the intrinsically disordered protein (IDP) FlgM and several macromolecular crowders we have revealed evidence for attraction between protein and crowder under cell-like conditions. The majority of biophysical studies use solutions that are dilute relative to in vivo conditions. By conducting studies in solutions at concentrations comparable to those of cellular environments, we may further our understanding of how proteins behave in the conditions in which they function. While lacking well-defined structures, IDPs are recognized as playing key roles in cellular signaling processes. FlgM is an IDP that acts as a negative regulator of flagellin synthesis. It is readily expressed and robust, making it an excellent system for studying the effects of crowding on IDPs. Via contrast matching, small-angle neutron scattering (SANS) allows isolation of the FlgM signal from that of the crowder. Perdeuterated FlgM was expressed and purified at Oak Ridge National Laboratory with SANS experiments conducted at the EQ-SANS diffractometer. Over a concentration range of 75-450 mg/mL, four crowders were used: dextran, Ficoll, bovine serum albumin, and lysozyme. Ensemble optimization modelling (EOM) was used to analyze the SANS profiles to gain information on the conformational ensembles of FlgM in dilute and crowded conditions. Analysis suggests that as crowder concentration increases, the average radius of gyration of FlgM initially decreases and subsequently increases. The latter is an indication of attraction between protein and crowder. Circular dichroism experiments showed an increase in helical content of FlgM as the concentration of dextran or Ficoll was increased. These studies, along with complementary NMR experiments, will allow us to quantitatively characterize the conformation and internal dynamics of FlgM in dilute and crowded conditions.

Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding

Event Abstract Back to Event Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding Valentina Magno1, Bella Roßbach2, Krithika Hariharan2, Jens Friedrichs1, Andreas Kurtz2 and Carsten Werner1, 3 1 Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Germany 2 Charité – Universitätsmedizin Berlin, Berlin-Brandenburg Center for Regenerative Therapies, Germany 3 Center for Regenerative Therapies Dresden, Technische Universität Dresden, Germany Introduction: Cell-derived extracellular matrices (ECMs) have shown in the latest years to have broad application in the field of tissue engineering and regenerative medicine. The recent introduction of macromolecular crowding (MMC) for the modulation of in vitro ECM production allowed, furthermore, to better mimic physiological microenvironments[1]. In this work, we aim to explore the effect of MMC on the secretion of ECM by primary mouse mesangial cells and to study the impact of decellularized ECMs produced under standard and MMC conditions on the growth and differentiation of human induced pluripotent stem cell (iPSC)-derived intermediate mesoderm (IM) progenitors[2]. Methods: Covalently attached mesangial cell-derived ECMs were produced on a maleic anhydride co-polymer platform[3]. The ECM secretion was carried out for 8 days either under standard (Std) or MMC culture conditions by supplementing the medium with the synthetic polysaccharide Ficoll. During the ECM deposition phase, cell viability was assessed using a PrestoBlue assay. Cell layers were decellularised using an hypotonic solution and DNA remnants were removed by DNase treatment. The impact of MMC on ECM composition and morphology was investigated by colorimetric assays, immunohistochemistry, atomic force microscopy and scanning electron microscopy. IM progenitors were seeded in parallel on the mesangial cell-derived ECMs as well as on Geltrex and collagen IV as control substrates, with or without the supplementation of the ROCK inhibitor Y-27632. Adhesion and proliferation of IM progenitors were analyzed respectively using a Crystal Violet and a PrestoBlue assay. Furthermore, the expression of relevant kidney markers was examined via real time PCR. Results and Discussion: PrestoBlue assay showed that MMC did not affect the viability of the mesangial cells during ECM deposition. MMC was found to influence the composition, the mechanical properties and the supramolecular assembly of the mesangial cell-derived ECM. IM progenitors showed significantly higher cell viability and adhesion on both types of ECM (Std and MMC) compared to Geltrex and collagen IV. Particularly, in presence of the ROCK inhibitor, a significantly higher adhesion was found on MMC-ECM compared to both Std-ECM and control substrates. Higher proliferation rates were observed on both types of ECM vs. the controls, with the MMC-ECM outperforming Std-ECM. A significant increase in the expression of some mesangial markers and other glomerular markers was found for IM progenitors cultivated on MMC-ECM vs. Std-ECM, with even less mesangial-like characteristics found on the control substrates. Conclusion: Our results indicate that matrix based signals support the differentiation of the IM progenitors toward the mesangial lineage without the addition of soluble cues. In the future, the combination of cell-derived ECM and MMC may be utilized to study kidney progenitor differentiation and to develop bioengineered kidney tissues. A systematic study on different crowder categories and concentrations may also allow to control progenitor differentiation to obtain a reliable cell source for regenerative approaches. This work was supported by EU Marie Curie NephroTools FP7 Initial Training Network (ITN)

Tuning knot abundance in semiflexible chains with crowders of different sizes: a Monte Carlo study of DNA chains.

We use stochastic simulation techniques to sample the conformational space of linear semiflexible polymers in a crowded medium and study how the knotting properties depend on the crowder size and concentration. The abundance of physical knots in the chains, which for definiteness we model on 10 kb long DNA filaments, is shown to have a non-monotonic, unimodal dependence on the colloid diameter, dc. The maximum incidence of knots occurs when dc is about equal to half of the gyration radius of the isolated chain. The degree of enhancement of knots grows rapidly with the solution density and can be very conspicuous relative to the case of isolated chains with no crowders. For instance, at 30% volume fraction the relative increase is more than fourfold. This dramatic enhancement is shown to originate from the depletion-induced chain compaction over multiple and concurring length scales. The same effect accounts for the variations of the knot length that accompany the changes in knotting probability. The findings suggest that crowded media could be viably used as a passive physical means for controlling and modulating the incidence and length of knots in DNA and other types of semiflexible polymers.

Do Macromolecular Crowding Agents Exert Only an Excluded Volume Effect? A Protein Solvation Study.

The effect of macromolecular crowding on protein structure and dynamics has mostly been explained on the basis of the excluded volume effect, its origin being entropic. In recent times a progressive shift in this view has been taking place with increasing emphasis on soft interactions that are enthalpic by nature. Using very low concentrations (1-10 g/L) of both synthetic (dextran- and poly(ethylene glycol) (PEG)-based) and protein (α-synuclein and myoglobin)-based crowders, we have shown that the solvation of probe molecule ANS (1-anilinonapthalene-8-sulfonate) bound to serum proteins bovine serum albumin (BSA) and human serum albumin (HSA) is significantly modulated in both a protein- and crowder-dependent fashion. Since under such conditions the effect of excluded volume is appreciably low, we propose that our observations are direct evidence of soft interactions between the macromolecular crowding agents used and the serum proteins. Moreover, our data reveal, that since at these low crowder concentrations major perturbations to the protein structure are unlikely to take place while minor perturbations might not be readily visible, protein solvation provides a unique spectral signature for capturing such local dynamics, thereby allowing one to decouple hard-sphere interactions from soft sphere ones. Furthermore, since fast fluctuations are known to play a major role in determining the functional characteristics of proteins and enzymes, our results suggest that such motions are prone to be modulated even when the cellular crowding conditions are quite relaxed. In other words, by the time the excluded volume effects come into the picture in the physiological milieu, modulations of functionally important protein motions that need a relatively lower activation energy have already taken place as a result of the aforementioned enthalpic (soft) interactions.

Crowding Induces Complex Ergodic Diffusion and Dynamic Elongation of Large DNA Molecules

Despite the ubiquity of molecular crowding in living cells, the effects of crowding on the dynamics of genome-sized DNA are poorly understood. Here, we track single, fluorescent-labeled large DNA molecules (11, 115 kbp) diffusing in dextran solutions that mimic intracellular crowding conditions (0–40%), and determine the effects of crowding on both DNA mobility and conformation. Both DNAs exhibit ergodic Brownian motion and comparable mobility reduction in all conditions; however, crowder size (10 vs. 500 kDa) plays a critical role in the underlying diffusive mechanisms and dependence on crowder concentration. Surprisingly, in 10-kDa dextran, crowder influence saturates at ∼20% with an ∼5× drop in DNA diffusion, in stark contrast to exponentially retarded mobility, coupled to weak anomalous subdiffusion, with increasing concentration of 500-kDa dextran. Both DNAs elongate into lower-entropy states (compared to random coil conformations) when crowded, with elongation states that are gamma distributed and fluctuate in time. However, the broadness of the distribution of states and the time-dependence and length scale of elongation length fluctuations depend on both DNA and crowder size with concentration having surprisingly little impact. Results collectively show that mobility reduction and coil elongation of large crowded DNAs are due to a complex interplay between entropic effects and crowder mobility. Although elongation and initial mobility retardation are driven by depletion interactions, subdiffusive dynamics, and the drastic exponential slowing of DNA, up to ∼300×, arise from the reduced mobility of larger crowders. Our results elucidate the highly important and widely debated effects of cellular crowding on genome-sized DNA.

Molecular crowding effect on dynamics of DNA-binding proteins search for their targets.

DNA-binding proteins locate and bind their target sequences positioned on DNA in crowded environments, but the molecular crowding effect on this search process is not clear. Using analytical techniques and Langevin dynamics simulations in two dimensions (2D), we find that the essential physics for facilitated diffusion in 2D search and 3D search is the same. We observe that the average search times have minima at the same optimal nonspecific binding energy for the cases with and without the crowding particle. Moreover, the molecular crowding increases the search time by increasing the average search rounds and the one-dimensional (1D) sliding time of a round, but almost not changing the average 2D diffusion time of a round. In addition, the fraction of 1D sliding time out of the total search time increases with increasing the concentration of crowders. For 2D diffusion, the molecular crowding decreases the jumping length and narrows its distribution due to the cage effect from crowders. These results shed light on the role of facilitated diffusion in DNA targeting kinetics in living cells.

Nonmonotonic diffusion of particles among larger attractive crowding spheres.

We study the diffusive motion of particles among fixed spherical crowders. The diffusers interact with the crowders through a combination of a hard-core repulsion and a short-range attraction. The long-time effective diffusion coefficient of the diffusers is found to depend nonmonotonically on the strength of their attraction to the crowders. That is, for a given concentration of crowders, a weak attraction to the crowders enhances diffusion. We show that this counterintuitive fact can be understood in terms of the mesoscopic excess chemical potential landscape experienced by the diffuser. The roughness of this excess chemical potential landscape quantitatively captures the nonmonotonic dependence of the diffusion rate on the strength of crowder-diffuser attraction; thus, it is a purely static predictor of dynamic behavior. The mesoscopic view given here provides a unified explanation for enhanced diffusion effects that have been found in various systems of technological and biological interest.

Diffusion of tagged particles in a crowded medium

The influence of crowding on the diffusion of tagged particles in a dense medium is investigated in the framework of a mean-field model, derived in the continuum limit from a microscopic stochastic process with exclusion. The probability distribution function of the tagged particles obeys to a nonlinear Smoluchowski equation, where the force and diffusion terms are determined self-consistently by the concentration of crowders in the medium. Transient sub-diffusive or super-diffusive behaviors are observed, depending on the selected initial conditions, that bridge normal diffusion regimes characterized by different diffusion coefficients. These anomalous crossovers originate from the microscopic competition for space and reflect the peculiar form of the non-homogeneous force term in the governing equation. Our results strongly warn against the overly simplistic identification of crowding with anomalous transport tout court.

Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Small-angle neutron scattering was used to study the effects of macromolecular crowding by two globular proteins, i.e., bovine pancreatic trypsin inhibitor and equine metmyoglobin, on the conformational ensemble of an intrinsically disordered protein, the N protein of bacteriophage λ. The λ N protein was uniformly labeled with 2H, and the concentrations of D2O in the samples were adjusted to match the neutron scattering contrast of the unlabeled crowding proteins, thereby masking their contribution to the scattering profiles. Scattering from the deuterated λ N was recorded for samples containing up to 0.12 g/mL bovine pancreatic trypsin inhibitor or 0.2 g/mL metmyoglobin. The radius of gyration of the uncrowded protein was estimated to be 30 Å and was found to be remarkably insensitive to the presence of crowders, varying by <2 Å for the highest crowder concentrations. The scattering profiles were also used to estimate the fractal dimension of λ N, which was found to be ∼1.8 in the absence or presence of crowders, indicative of a well-solvated and expanded random coil under all of the conditions examined. These results are contrary to the predictions of theoretical treatments and previous experimental studies demonstrating compaction of unfolded proteins by crowding with polymers such as dextran and Ficoll. A computational simulation suggests that some previous treatments may have overestimated the effective volumes of disordered proteins and the variation of these volumes within an ensemble. The apparent insensitivity of λ N to crowding may also be due in part to weak attractive interactions with the crowding proteins, which may compensate for the effects of steric exclusion.

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg(2+) ion concentrations are low, K(+) concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg(2+) (0.5-2 mM) and K(+) (140 mM) if the solution is supplemented with physiological amounts (∼ 20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.

Evaluation of Proteins' Rotational Diffusion Coefficients from Simulations of Their Free Brownian Motion in Volume-Occupied Environments.

We have investigated the rotational dynamics of hen egg white lysozyme in monodisperse aqueous solutions of concentrations up to 250 mg/mL, using a rigid-body Brownian dynamics method that accurately accounts for anisotropies of diffusing objects. We have examined the validity of the free diffusion concept in the analysis of computer simulations of volume-occupied molecular solutions. We have found that, when as the only intermolecular interaction, the excluded volume effect is considered, rotational diffusion of molecules adheres to the free diffusion model. Further, we present a method based on the exact (in the case of the free diffusion) analytic forms of autocorrelation functions of particular vectors rigidly attached to diffusing objects, which allows one to obtain from results of molecular simulations the three principal rotational diffusion coefficients characterizing rotational Brownian motion of an arbitrarily shaped rigid particle for an arbitrary concentration of crowders. We have applied this approach to trajectories resulting from Brownian dynamics simulations of hen egg white lysozyme solutions. We show that the apparent anisotropy of proteins' rotational motions increases with an increasing degree of crowding. Finally, we demonstrate that even if the hydrodynamic anisotropy of molecules is neglected and molecules are simulated using their average translational and rotational diffusion coefficients, excluded volume effects still lead to their anisotropic rotational dynamics.

Macromolecular crowding as a suppressor of human IAPP fibril formation and cytotoxicity.

The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most in vitro studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a – consequently - retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.

Dual effect of crowders on fibrillation kinetics of polypeptide chains revealed by lattice models

We have developed the lattice model for describing polypeptide chains in the presence of crowders. The influence of crowding confinement on the fibrillation kinetics of polypeptide chains is studied using this model. We observed the non-trivial behavior of the fibril formation time τfib that it decreases with the concentration of crowders if crowder sizes are large enough, but the growth is observed for crowders of small sizes. This allows us to explain the recent experimental observation on the dual effect of crowding particles on fibril growth of proteins that for a fixed crowder concentration the fibrillation kinetics is fastest at intermediate values of total surface of crowders. It becomes slow at either small or large coverages of cosolutes. It is shown that due to competition between the energetics and entropic effects, the dependence of τfib on the size of confined space is described by a parabolic function.

Reduced Native State Stability in Crowded Cellular Environment Due to Protein–Protein Interactions

The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects.

Macromolecular Crowding Stabilizes the Functional, Non-Toxic State of IAPP by Suppressing its Fibrillation

The interior of the biological cell is known to be a crowded milieu, which significantly influences protein association and aggregation. As several cell degenerative diseases, such as Parkinson¯s disease or type-2 diabetes mellitus, are related to the misfolding, self-association and subsequent fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. This study focuses on the properties of human islet amyloid polypeptide (hIAPP) in crowded environments of two different kinds: network-like structures formed by polysaccharides and high concentrations of inert globular proteins. Two distinct processes could be distinguished in these crowded solutions: The formation of stable globular off-pathway species, and the usual hIAPP aggregation pathway from a disordered monomeric structure via nuclei formation to fibril formation. To which extent the different pathways are populated is shown to depend markedly on the crowder concentration and the geometry of the confinement. Different to other amyloidogenic peptides, the latter process is retarded or even inhibited at high crowding concentrations, but unchanged on the mechanistic level. As hIAPP is related to type-2 diabetes mellitus and presumably responsible for the disease accompanying β-cell-membrane permeabilization and the final β-cell loss, hIAPP specific cytotoxicity assays were conducted as well. Conversely to the high cytotoxicity exhibited by the normal fibrillation pathway, the data reveal a non-toxic effect for the off-pathway species stabilized through the crowding agents. From these results it can be postulated that cellular crowding is able to stabilize the native, non-toxic and functional conformation of hIAPP inside the biological cell.

Crowding Effects on the Formation and Maintenance of Nuclear Bodies: Insights from Molecular-Dynamics Simulations of Simple Spherical Model Particles

The physics of structure formation and maintenance of nuclear bodies (NBs), such as nucleoli, Cajal bodies, promyelocytic leukemia bodies, and speckles, in a crowded nuclear environment remains largely unknown. We investigate the role of macromolecular crowding in the formation and maintenance of NBs using computer simulations of a simple spherical model, called Lennard-Jones (LJ) particles. LJ particles form a one-phase, dilute fluid when the intermolecular interaction is weaker than a critical value, above which they phase separate and form a condensed domain. We find that when volume-exclusive crowders exist in significant concentrations, domain formation is induced even for weaker intermolecular interactions, and the effect is more pronounced with increasing crowder concentration. Simulation results show that a previous experimental finding that promyelocytic leukemia bodies disappear in the less-crowded condition and reassemble in the normal crowded condition can be interpreted as a consequence of the increased intermolecular interactions between NB proteins due to crowding. Based on further analysis of the simulation results, we discuss the acceleration of macromolecular associations that occur within NBs, and the delay of diffusive transport of macromolecules within and out of NBs when the crowder concentration increases. This study suggests that in a polydisperse nuclear environment that is enriched with a variety of macromolecules, macromolecular crowding not only plays an important role in the formation and maintenance of NBs, but also may perform some regulatory functions in response to alterations in the crowding conditions.

Determining the Optimal Degree of Macromolecular Crowding in Solution Using Fluorescence Recovery after Photobleaching (FRAP)

Macromolecular crowding (MMC) is a feature of cellular interiors and, in multicellular organisms, also of extracellular environments. Recreating this crowdedness via employment of macromolecules is a valuable tool in studying protein folding behaviour, supramolecular assembly, acceleration of biochemical reactions and extracellular matrix deposition. Optimal MMC in solution has been mostly determined empirically by quantitating biochemical endpoints (PCR product, amount of matrix deposited). Here, we sought to develop a technique allowing the direct observation of MMC in a purely physical manner in order to make predictions of optimal crowder concentrations. We have shown earlier by dynamic light scattering that above certain concentrations the hydrodynamic radii of Ficolls 70 and 400 shrink (Harve et al 2007). We described this event as self-crowding.Here, we studied this phenomenon with FRAP in order to monitor changes in the molecular diffusion velocity of FITC-tagged Ficoll 70 and Ficoll 400. Both crowders are sucrose polymers, stable at neutral pH, and globular in shape.FRAP allowed us to establish a clear link between molecular speed variation and degree of crowdedness. We verified that beyond a threshold concentration self-crowding occurred as evidenced by a speed increase for the molecules undergoing shrinking. Maximum compaction of Ficoll molecules was reached with increasing concentrations. Further concentration increases led to a rapid drop of diffusion velocity.By purely physical means we can now define the optimal crowding concentration as the greatest possible concentration allowing for the greatest possible diffusion velocity. Interestingly enough, the determined values match those obtained from biological read-outs published earlier (Chen et al 2011) very well. This technique now opens up the possibility to determine for each potential macromolecule the crowding points rapidly, and to predict suitable concentrations for application in biological systems.

Quantitative Characterization of Temperature-Independent and Temperature-Dependent Protein–Protein Interactions in Highly Nonideal Solutions

The interaction among each of three dilute "tracer" proteins (bovine serum albumin, superoxide dismutase, and ovomucoid) at a concentration of 2 mg/mL and each of two "crowder" proteins (ovomucoid and BSA) at concentrations up to 100 mg/mL was characterized by analysis of dependence of the equilibrium gradients of both tracer and crowder upon the concentration of crowder. The equilibrium gradients of both crowder proteins were found to be independent of temperature over the range 5-37 °C. The equilibrium gradients of tracer BSA and ovomucoid in the complementary crowder species were likewise found to be independent of temperature over this range, indicating that interaction among these tracers and crowders is predominantly repulsive and essentially entirely entropic in nature. The equilibrium gradient of tracer SOD in BSA was also found to be independent of temperature over this range, but the gradient of tracer SOD in ovomucoid depended significantly upon temperature in a manner indicating a significant enthalpic (attractive) component of the overall interaction between SOD and ovomucoid. The experimental data are analyzed using model-free expansions of the thermodynamic activity coefficients of tracer and crowder in powers of the concentration of crowder and using approximate statistical thermodynamic models based upon highly simplified descriptions of molecular structure and interactions. Detailed analysis of the results indicates a relatively small contribution of nonspecific attraction to the total protein-protein interaction, which is dominated by steric repulsion.