Excluded‐Volume Effects in Living Cells

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in‐cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET‐labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded‐volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in‐cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in‐cell crowding effect as a modulator of biomolecular reactions.     

Inactivation of Recombinant Human Brain-Type Creatine Kinase During Denaturation by Guanidine Hydrochloride in a Macromolecular Crowding System

In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k ₂ value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8?×?10^?5?s^?1. The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment.     

Folding dynamics of Trp-cage in the presence of chemical interference and macromolecular crowding. I

Proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been well established that the so-called "macromolecular crowding" effect enhances the folding stability of proteins by destabilizing their unfolded states for selected proteins. On the other hand, chemical and thermal denaturation is often used in experiments as a tool to destabilize a protein by populating the unfolded states when probing its folding landscape and thermodynamic properties. However, little is known about the complicated effects of these synergistic perturbations acting on the kinetic properties of proteins, particularly when large structural fluctuations, such as protein folding, have been involved. In this study, we have first investigated the folding mechanism of Trp-cage dependent on urea concentration by coarse-grained molecular simulations where the impact of urea is implemented into an energy function of the side chain and/or backbone interactions derived from the all-atomistic molecular dynamics simulations with urea through a Boltzmann inversion method. In urea solution, the folding rates of a model miniprotein Trp-cage decrease and the folded state slightly swells due to a lack of contact formation between side chains at the terminal regions. In addition, the equilibrium m-values of Trp-cage from the computer simulations are in agreement with experimental measurements. We have further investigated the combined effects of urea denaturation and macromolecular crowding on Trp-cage's folding mechanism where crowding agents are modeled as hard-spheres. The enhancement of folding rates of Trp-cage is most pronounced by macromolecular crowding effect when the extended conformations of Trp-cast dominate at high urea concentration. Our study makes quantitatively testable predictions on protein folding dynamics in a complex environment involving both chemical denaturation and macromolecular crowding effects.     

大分子拥挤条件下光诱导辣根过氧化物酶还原的光谱研究

稀溶液中光诱导辣根过氧化物酶(Horseradish Peroxidase, HRP)能够发生还原反应,但这一研究忽略了生物体细胞内因存在各种分子而高度拥挤的真实环境。本文采用紫外可见、荧光、同步荧光及圆二色光谱法,研究了大分子拥挤环境中光诱导HRP的还原过程及外界环境对其光还原的影响。UV-Vis光谱和同步荧光光谱结果表明,采用403 nm单色光照射时,HRP在拥挤试剂葡聚糖70(Dextran70)中还原程度较好,而HRP在聚蔗糖70(Ficoll70)拥挤环境下不发生光还原反应;采用280 nm单色光照射时,在拥挤试剂Dextran70和Ficoll70中HRP光还原反应程度增加,且HRP的光还原程度在Ficoll70溶液中远高于在Dextran70溶液中;光诱导HRP还原的最适温度在Dextran70和Ficoll70环境中分别为4 ℃和24 ℃;HRP在Dextran70溶液中的最佳光还原浓度是100 g/L,在Ficoll70环境中HRP还原程度随着Ficoll70浓度的增加而增大;CD光谱结果表明拥挤环境中HRP被光还原后,蛋白的二级结构基本没有变化。     

Suppression of linear side products by macromolecular crowding in nonribosomal enterobactin biosynthesis

Nonribosomal enterobactin synthetase of Escherichia coli was found to prematurely release a large amount of linear precursors in an in vitro reconstitution. However, these side products are suppressed to negligible levels by polymeric cosolvents that create macromolecular crowding, a prominent feature of the intracellular environment. These findings show that macromolecular crowding is essential to normal functioning of the nonribosomal peptide synthetase and suggest that it may be crucial to biotechnological utilization of similar enzyme systems.     

Enzyme Entrapped in Polymer‐Modified Nanopores: The Effects of Macromolecular Crowding and Surface Hydrophobicity

Macromolecular crowding is an ubiquitous phenomenon in living cells that significantly affects the function of enzymes in vivo. However, this effect has not been paid much attention in the research of the immobilization of enzymes onto mesoporous silica. Herein, we report the combined effects of macromolecular crowding and surface hydrophobicity on the performance of an immobilized enzyme by accommodating lipase molecules into a series of mesoporous silicas with different amounts of inert poly(methacrylate) (PMA) covalently anchored inside the nanopores. The incorporation of the PMA polymer into the nanopores of mesoporous silica enables the fabrication of a crowded and hydrophobic microenvironment for the immobilized enzyme and the variation in polymer content facilitates an adjustment of the degree of crowding and surface properties of this environment. Based on this system, the catalytic features of immobilized lipase were investigated as a function of polymer content in nanopores and the results indicated that the catalytic efficiency, thermostability, and reusability of immobilized lipase could all be improved by taking advantage of the macromolecular crowding effect and surface hydrophobicity. These findings provide insight into the possible functions of the macromolecular crowding effect, which should be considered and integrated into the fabrication of suitable mesoporous silicas to improve enzyme immobilization.     

Effects of quenched and annealed macromolecular crowding elements on a simple model for signaling in T lymphocytes

Biochemical reactions in cells occur in an environment that is crowded in the sense that various macromolecular species and organelles occupy much of the space. The effects of molecular crowding on biochemical reactions have usually been studied in the past in a spatially homogeneous environment. However, signal transduction in cells is often initiated by the binding of receptors and ligands in two apposed cell membranes, and the pertinent biochemical reactions occur in a spatially inhomogeneous environment. We have studied the effects of crowding on biochemical reactions that involve both membrane proteins and cytosolic molecules by investigating a simplified version of signaling in T lymphocytes using a Monte Carlo algorithm. We find that, if signal transduction occurs on time scales that are slow compared to the motility of the molecules and organelles that constitute the crowding elements, the effects of crowding are qualitatively the same as in a homogeneous three-dimensional (3D) medium. In contrast, if signal transduction occurs on a time scale that is much faster than the time over which the crowding elements move, then the effects of varying the extent of crowding are very different when reactions occur in both 2- and 3D space. We discuss these differences and their origin. Since many signaling reactions are fast, our results may be useful for diverse situations in cell biology.     

Residue-level interrogation of macromolecular crowding effects on protein stability

Theory predicts that macromolecular crowding affects protein behavior, but experimental confirmation is scant. Herein, we report the first residue-level interrogation of the effects of macromolecular crowding on protein stability. We observe up to a 100-fold increase in the stability, as measured by the equilibrium constant for folding, for the globular protein chymotrypsin inhibitor 2 (CI2) in concentrations of the cosolute poly(vinylpyrrolidone) (PVP) that mimic the protein concentration in cells. We show that the increased stability is caused by the polymeric nature of PVP and that the degree of stabilization depends on both the location of the individual residue in the protein structure and the PVP concentration. Our data reinforce the assertion that macromolecular crowding stabilizes the protein by destabilizing its unfolded states.     

Factors controlling the mobility of photosynthetic proteins

Protein diffusion in and around the photosynthetic membrane must play a crucial role in photosynthetic functions including electron transport, regulation of light-harvesting, and biogenesis, turnover and repair of membrane components. Protein mobility is controlled by a complex web of specific interactions, plus the viscosity of the environment and the extent of macromolecular crowding. I discuss the techniques that can be used to measure protein mobility in photosynthetic membranes. I then summarize what we know about the constraints on protein mobility imposed by macromolecular aggregation and crowding in and around the thylakoid membranes of green plants and cyanobacteria, with particular reference to the fluidity of the thylakoid membrane and the aqueous phases on either side of the membrane (the stroma/cytoplasm and the thylakoid lumen). Current indications are that the stroma/cytoplasm is a relatively fluid environment, whereas protein mobility in the lumen may be extremely restricted. The thylakoid membrane itself has an intermediate fluidity: some protein complexes are virtually immobile, probably due to their incorporation into large, stable macromolecular aggregates. However, there is sufficient free space to allow the long-range diffusion of some complexes. Finally, I discuss some future directions for research in this area.     

15N NMR spin relaxation dispersion study of the molecular crowding effects on protein folding under native conditions

The effects of macromolecular crowding on protein stability and folding kinetics have been studied using the recently developed 15N spin relaxation dispersion technique. By applying this method to a redesigned apocytochrome b562, the kinetics and thermodynamics of the protein folding processes in both the presence and the absence of crowding agents have been characterized. The result indicates that, even under the mild crowded environments (in the presence of 85 mg/mL of PEG 20K), the folding rate of the protein can speed up significantly while the unfolded rate remains unchanged within experimental error.     

Effect of macromolecular crowding on DNA:Au nanoparticle bioconjugate assembly

DNA:Au nanosphere bioconjugates have applications in biosensing and in the bottom-up assembly of materials. These bioconjugates can be selectively assembled into three-dimensional aggregates upon addition of complementary DNA oligonucleotides and can be dissociated by heating above a melting transition temperature at which the DNA duplexes are denatured. Herein we describe the impact of polymeric solutes on the thermal denaturation behavior of DNA:Au nanoparticle bioconjugate assemblies. Polymeric solutes can dramatically impact biochemical reactions via macromolecular crowding. Poly(ethylene glycol)s (PEGs) and dextrans of varying molecular weights were used as crowding reagents. While both PEG and dextran increased the stability of DNA:Au aggregates, melting transition temperatures in the presence of PEG were impacted more significantly. Polymer molecular weight was less important than polymer chemistry and weight percent in solution. For a high (15%) weight percent of PEG, aggregation was observed even in the absence of complementary oligonucleotides. These results underscore the importance of polymer chemistry in addition to physical volume exclusion in macromolecular crowding and point to the importance of understanding these effects when designing biorecognition-based nanoparticle assembly schemes in complex matrixes (i.e., any involving polymeric solutes).     

The role of macromolecular crowding in single-entity electrochemistry: Friend or foe?

The recent development of nanoscale probes has enabled the study of single molecules and single cells with unprecedented resolution and the expansion of the field of single-entity electrochemistry. There is a growing evidence suggesting that highly crowded intracellular environment facilitate nanoelectrochemical measurements in cells by improving the signal-to-noise ratio. In this opinion piece, we discuss the concept of macromolecular crowding and its implications in single-entity electrochemistry.     

Toward realistic modeling of dynamic processes in cell signaling: quantification of macromolecular crowding effects

One of the major factors distinguishing molecular processes in vivo from biochemical experiments in vitro is the effect of the environment produced by macromolecular crowding in the cell. To achieve a realistic modeling of processes in the living cell based on biochemical data, it becomes necessary, therefore, to consider such effects. We describe a protocol based on Brownian dynamics simulation to characterize and quantify the effect of various forms of crowding on diffusion and bimolecular association in a simple model of interacting hard spheres. We show that by combining the elastic collision method for hard spheres and the mean field approach for hydrodynamic interaction (HI), our simulations capture the correct dynamics of a monodisperse system. The contributions from excluded volume effect and HI to the crowding effect are thus quantified. The dependence of the results on size distribution of each component in the system is illustrated, and the approach is applied as well to the crowding effect on electrostatic-driven association in both neutral and charged environments; values for effective diffusion constants and association rates are obtained for the specific conditions. The results from our simulation approach can be used to improve the modeling of cell signaling processes without additional computational burdens.     

The influence of macromolecular crowding on HIV-1 protease internal dynamics

High macromolecular concentrations, or crowded conditions, have been shown to affect a wide variety of molecular processes, including diffusion, association and dissociation, and protein folding and stability. Here, we model the effect of macromolecular crowding on the internal dynamics of a protein, HIV-1 protease, using Brownian dynamics simulations. HIV-1 protease possesses a pair of flaps which are postulated to open in the early stages of its catalytic mechanism. Compared to low concentrations, close-packed concentrations of repulsive crowding agents are found to significantly reduce the fraction of time that the protease flaps are open. Macromolecular crowding is likely to have a major effect on in vivo enzyme activity, and may play an important regulatory role in the viral life cycle.     

酸诱导拥挤条件下肌红蛋白及突变体去折叠过程

采用紫外-可见吸收光谱、同步荧光光谱和圆二色(CD)光谱法研究拥挤试剂葡聚糖70 (Dextran70)和聚蔗糖70 (Ficoll70)存在条件下, 酸诱导野生型肌红蛋白Mb(WT)及其突变体Mb(D60K)的去折叠过程. 结果显示: 在Dextran70 和Ficoll70 两种拥挤环境下, Mb(WT)的变性中点pH值由4.25 分别降低到3.78 与3.76, 拥挤试剂加入后增强了Mb(WT)的耐酸能力; 肌红蛋白60位天冬氨酸(Asp)突变为赖氨酸(Lys)后, 变性中点pH值由4.25 降低到4.19, 耐酸性比野生型肌红蛋白有所增强, Mb(D60K)在Dextran70 和Ficoll70 两种拥挤环境下变性中点pH值由4.19 分别降至3.74 和3.12. 以上实验说明肌红蛋白表面氨基酸突变和拥挤试剂的添加起到了稳定血红素微环境、芳香族氨基酸及二级结构和保护蛋白天然状态的作用.     

Macromolecular crowding improves polymer encapsulation within giant lipid vesicles

We report the effect of macromolecular crowding on encapsulation efficiency of fluorescently labeled poly(ethylene glycol) (PEG) and dextran polymers within individual giant lipid vesicles (GVs). Low concentrations of the fluorescently labeled polymers (82 nM to 186 pM) were mixed with varying concentrations of nonfluorescent polymers that served as crowding agents during vesicle formation by gentle hydration. Encapsulation efficiency of the fluorescently labeled polymers in individual GVs (EEind) was determined via confocal fluorescence microscopy. EEind for high molecular weight polymers (e.g., fluorescein isothiocyanate (FITC)-dextran 500 and 2000 kDa) increased substantially in the presence of several weight percent unlabeled PEG or dextran. For example, when 0.24 microM FITC dextran 500 kDa was encapsulated, addition of 3% PEG 8 kDa improved the mean concentration in the GVs from 0.14 microM (+/-50%) to 0.24 microM (+/-12%). Light scattering data indicate reduced hydrodynamic radii for polymers as a function of increasing polymer concentration, suggesting that the improvements in EEind result from polymer condensation due to macromolecular crowding. Polymeric cosolutes did not significantly impact EEind for lower molecular weight polymers (e.g., Alexa Fluor 488-PEG 20 kDa), which already encapsulated efficiently (EEind to approximately 1). However, for both the higher and lower molecular weight labeled polymers, cosolutes led to improved uniformity in EEind for vesicles within a batch. Methods for improving the value and homogeneity of EEind for polymeric solutes in lipid vesicles are important in a variety of applications, including the use of vesicles as microreactors and as vehicles for drug delivery.     

Translational Dynamics of Lipidated Ras Proteins in the Presence of Crowding Agents and Compatible Osmolytes

Ras proteins are small GTPases and are involved in transmitting signals that control cell growth, differentiation, and proliferation. Since the cell cytoplasm is crowded with different macromolecules, understanding the translational dynamics of Ras proteins in crowded environments is crucial to yielding deeper insight into their reactivity and function. Herein, the translational dynamics of lipidated N‐Ras and K‐Ras4B is studied in the bulk and in the presence of a macromolecular crowder (Ficoll) and the compatible osmolyte and microcrowder sucrose by fluorescence correlation spectroscopy. The results reveal that N‐Ras forms dimers due to the presence of its lipid moiety in the hypervariable region, whereas K‐Ras4B remains in its monomeric form in the bulk. Addition of a macromolecular crowding agent gradually favors clustering of the Ras proteins. In 20 wt % Ficoll N‐Ras forms trimers and K‐Ras4B dimers. Concentrations of sucrose up to 10 wt % foster formation of N‐Ras trimers and K‐Ras dimers as well. The results can be rationalized in terms of the excluded‐volume effect, which enhances the association of the proteins, and, for the higher concentrations, by limited‐hydration conditions. The results of this study shed new light on the association state of these proteins in a crowded environment. This is of particular interest for the Ras proteins, because their solution state—monomeric or clustered—influences their membrane‐partitioning behavior and their interplay with cytosolic interaction partners.     

Effect of Molecular Crowding on the Temperature–Pressure Stability Diagram of Ribonuclease A

FT‐IR spectroscopic and thermodynamic measurements were designed to explore the effect of a macromolecular crowder, dextran, on the temperature and pressure‐dependent phase diagram of the protein Ribonuclease A (RNase A), and we compare the experimental data with approximate theoretical predictions based on configuration entropy. Exploring the crowding effect on the pressure‐induced unfolding of proteins provides insight in protein stability and folding under cell‐like dense conditions, since pressure is a fundamental thermodynamic variable linked to molecular volume. Moreover, these studies are of relevance for understanding protein stability in deep‐sea organisms, which have to cope with pressures in the kbar range. We found that not only temperature‐induced equilibrium unfolding of RNase A, but also unfolding induced by pressure is markedly prohibited in the crowded dextran solutions, suggesting that crowded environments such as those found intracellularly, will also oppress high‐pressure protein unfolding. The FT‐IR spectroscopic measurements revealed a marked increase in unfolding pressure of 2 kbar in the presence of 30 wt % dextran. Whereas the structural changes upon thermal unfolding of the protein are not significantly influenced in the presence of the crowding agent, through stabilization by dextran the pressure‐unfolded state of the protein retains more ordered secondary structure elements, which seems to be a manifestation of the entropic destabilization of the unfolded state by crowding.     

Cost‐effective imprinting combining macromolecular crowding and a dummy template for the fast purification of punicalagin from pomegranate husk extract

The combination of molecular crowding and virtual imprinting was employed to develop a cost‐effective method to prepare molecularly imprinted polymers. By using linear polymer polystyrene as a macromolecular crowding agent, an imprinted polymer recognizable to punicalagin had been successfully synthesized with punicalin as the dummy template. The resulting punicalin‐imprinted polymer presented a remarkable selectivity to punicalagin with an imprinting factor of 3.17 even at extremely low consumption of the template (template/monomer ratio of 1:782). In contrast, the imprinted polymer synthesized without crowding agent, did not show any imprinting effect at so low template amount. The imprinted polymers made by combination of molecular crowding and virtual imprinting can be utilized for the fast separation of punicalagin from pomegranate husk extract after optimizing the protocol of solid‐phase extraction with the recovery of 85.3 ± 1.2%.