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1.  Effects of Macromolecular Crowding on Protein Conformational Changes 
PLoS Computational Biology  2010;6(7):e1000833.
Many protein functions can be directly linked to conformational changes. Inside cells, the equilibria and transition rates between different conformations may be affected by macromolecular crowding. We have recently developed a new approach for modeling crowding effects, which enables an atomistic representation of “test” proteins. Here this approach is applied to study how crowding affects the equilibria and transition rates between open and closed conformations of seven proteins: yeast protein disulfide isomerase (yPDI), adenylate kinase (AdK), orotidine phosphate decarboxylase (ODCase), Trp repressor (TrpR), hemoglobin, DNA β-glucosyltransferase, and Ap4A hydrolase. For each protein, molecular dynamics simulations of the open and closed states are separately run. Representative open and closed conformations are then used to calculate the crowding-induced changes in chemical potential for the two states. The difference in chemical-potential change between the two states finally predicts the effects of crowding on the population ratio of the two states. Crowding is found to reduce the open population to various extents. In the presence of crowders with a 15 Å radius and occupying 35% of volume, the open-to-closed population ratios of yPDI, AdK, ODCase and TrpR are reduced by 79%, 78%, 62% and 55%, respectively. The reductions for the remaining three proteins are 20–44%. As expected, the four proteins experiencing the stronger crowding effects are those with larger conformational changes between open and closed states (e.g., as measured by the change in radius of gyration). Larger proteins also tend to experience stronger crowding effects than smaller ones [e.g., comparing yPDI (480 residues) and TrpR (98 residues)]. The potentials of mean force along the open-closed reaction coordinate of apo and ligand-bound ODCase are altered by crowding, suggesting that transition rates are also affected. These quantitative results and qualitative trends will serve as valuable guides for expected crowding effects on protein conformation changes inside cells.
Author Summary
The biophysical properties of proteins inside cells can be expected to be quite different from those typically measured by in vitro experiments in dilute solutions. In particular, intracellular macromolecular crowding may significantly affect the equilibria and transition rates between different conformations of a protein, and hence its functions. What are the trends and magnitudes of such crowding effects? We address this question here by applying a recently developed approach for modeling crowding. Seven proteins, each with structures for both an open state and a closed state, are studied. Crowding exerts significant effects on the open-closed equilibria of four proteins and more modest effects on the remaining three. Potentials of mean force along the open-closed reaction coordinate, and hence transition rates, are similarly affected. The extent of conformational changes is the main determinant for the magnitudes of crowding effects, but the protein size also plays an important role. The effects of crowding become stronger as the protein size increases. Conformational transitions of the ribosome, an extremely large complex, during translation are predicted to experience particularly strong effects of intracellular crowding. We conclude that deduction of intracellular behaviors from in vitro experiments requires explicit consideration of crowding effects.
PMCID: PMC2895631  PMID: 20617196
2.  The Effect of Macromolecular Crowding on the Electrostatic Component of Barnase–Barstar Binding: A Computational, Implicit Solvent-Based Study 
PLoS ONE  2014;9(6):e98618.
Macromolecular crowding within the cell can impact both protein folding and binding. Earlier models of cellular crowding focused on the excluded volume, entropic effect of crowding agents, which generally favors compact protein states. Recently, other effects of crowding have been explored, including enthalpically-related crowder–protein interactions and changes in solvation properties. In this work, we explore the effects of macromolecular crowding on the electrostatic desolvation and solvent-screened interaction components of protein–protein binding. Our simple model enables us to focus exclusively on the electrostatic effects of water depletion on protein binding due to crowding, providing us with the ability to systematically analyze and quantify these potentially intuitive effects. We use the barnase–barstar complex as a model system and randomly placed, uncharged spheres within implicit solvent to model crowding in an aqueous environment. On average, we find that the desolvation free energy penalties incurred by partners upon binding are lowered in a crowded environment and solvent-screened interactions are amplified. At a constant crowder density (fraction of total available volume occupied by crowders), this effect generally increases as the radius of model crowders decreases, but the strength and nature of this trend can depend on the water probe radius used to generate the molecular surface in the continuum model. In general, there is huge variation in desolvation penalties as a function of the random crowder positions. Results with explicit model crowders can be qualitatively similar to those using a lowered “effective” solvent dielectric to account for crowding, although the “best” effective dielectric constant will likely depend on multiple system properties. Taken together, this work systematically demonstrates, quantifies, and analyzes qualitative intuition-based insights into the effects of water depletion due to crowding on the electrostatic component of protein binding, and it provides an initial framework for future analyses.
PMCID: PMC4051634  PMID: 24915485
3.  Macromolecular Crowding as a Suppressor of Human IAPP Fibril Formation and Cytotoxicity 
PLoS ONE  2013;8(7):e69652.
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.
PMCID: PMC3726762  PMID: 23922768
4.  Volume Exclusion and Soft Interaction Effects on Protein Stability under Crowded Conditions† 
Biochemistry  2010;49(33):6984-6991.
Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams per liter of macromolecules often interfere with observing the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide 1H exchange. We analyze the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues where stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions need to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.
PMCID: PMC2927838  PMID: 20672856
5.  Influence of Nanoparticle Size and Shape on Oligomer Formation of an Amyloidogenic Peptide 
Understanding the influence of macromolecular crowding and nanoparticles on the formation of in-register β-sheets, the primary structural component of amyloid fibrils, is a first step towards describing in vivo protein aggregation and interactions between synthetic materials and proteins. Using all atom molecular simulations in implicit solvent we illustrate the effects of nanoparticle size, shape, and volume fraction on oligomer formation of an amyloidogenic peptide from the transthyretin protein. Surprisingly, we find that inert spherical crowding particles destabilize in-register β-sheets formed by dimers while stabilizing β-sheets comprised of trimers and tetramers. As the radius of the nanoparticle increases crowding effects decrease, implying smaller crowding particles have the largest influence on the earliest amyloid species. We explain these results using a theory based on the depletion effect. Finally, we show that spherocylindrical crowders destabilize the ordered β-sheet dimer to a greater extent than spherical crowders, which underscores the influence of nanoparticle shape on protein aggregation.
PMCID: PMC3117580  PMID: 21691423
crowding; in vivo; early events; amyloid
6.  Conformational Sampling of Peptides in the Presence of Protein Crowders from AA/CG-Multiscale Simulations 
The Journal of Physical Chemistry. B  2012;116(29):8610-8620.
Macromolecular crowding is recognized as an important factor influencing folding and conformational dynamics of proteins and nucleic acids. Previous views of crowding have focused on the mostly entropic volume exclusion effect of crowding but recent studies are indicating the importance of enthalpic effects, in particular changes in electrostatic interactions due to crowding. Here, temperature replica exchange molecular dynamics simulations of trp-cage and melittin in the presence of explicit protein crowders are presented to further examine the effect of protein crowders on peptide dynamics. The simulations involve a three-component multiscale modeling scheme where the peptides are represented at atomistic level, the crowder proteins at a coarse-grained level, and the surrounding aqueous solvent as implicit solvent. This scheme optimally balances a physically realistic description for the peptide with computational efficiency. The multiscale simulations were compared with simulations of the same peptides in different dielectric environments with dielectric constants ranging from 5 to 80. It is found that the sampling in the presence of the crowders resembles sampling with reduced dielectric constants between 10 and 40. Furthermore, diverse conformational ensembles are generated in the presence of crowders including partially unfolded states for trp-cage. These findings emphasize the importance of enthalpic interactions over volume exclusion effects in describing the effects of cellular crowding.
PMCID: PMC3398202  PMID: 22429139
protein-protein interactions; molecular dynamics; coarse-graining; multiscale simulations; replica exchange; crowding
7.  Macromolecular crowding induces polypeptide compaction and decreases folding cooperativity 
A cell's interior is comprised of macromolecules that can occupy up to 40% of its available volume. Such crowded environments can influence the stability of proteins and their rates of reaction. Using discrete molecular dynamics simulations, we investigate how both the size and number of neighboring crowding reagents affect the thermodynamic and folding properties of structurally diverse proteins. We find that crowding induces higher compaction of proteins. We also find that folding becomes less cooperative with the introduction of crowders into the system. The crowders may induce alternative non-native protein conformations, thus creating barriers for protein folding in highly crowded media.
PMCID: PMC3050011  PMID: 20355290
8.  Viscoelastic behavior and in vivo release of microgel dispersions with inverse thermo-reversible gelation 
Biomacromolecules  2007;9(1):142-148.
Microgels with two interpenetrating polymer networks of poly-N-isopropylacrylamide and poly-acrylic acid (PNIPAM-IPN-PAAc) were synthesized using a seed method. The IPN microgels in water have an average hydrodynamic radius of about 85 nm at 21 °C, measured by dynamic light scattering method. The atomic force microscope image showed that the particles were much smaller after they were dried but remain their spherical shape. The storage and loss moduli G' and G' ' of dispersions of IPN microgels were measured in the linear stress regime as functions of temperature and frequency at various polymer concentrations using a stress-controlled rheometer. For dispersions with high polymer concentration (3.5 and 6.0 wt%) and at high temperatures (34 and 38 °C), the samples behave as viscoelastic solids and the storage modulus was larger than the loss modulus over the entire frequency range. The loss tangent was measured at various frequencies as a function of temperature. The gelation temperature was determined to be 33 °C at the point where a frequency-independent value of the loss tangent was first observed.
Using an animal implantation model, the biocompatibility and drug release properties of the IPN microgl dispersion were evaluated. Fluorescein as a model drug was mixed into an aqueous microgel dispersion at ambient temperature. This drug loaded liquid was then injected subcutaneously in Balb/C mice from Taconic Farms. The test results have shown that the IPN microgels were biocompatible in this acute implantation model and the presence of gelled microgel dispersion substantially slowed the release of fluorescein.
PMCID: PMC3536501  PMID: 18067257
9.  A method for computing association rate constants of atomistically represented proteins under macromolecular crowding 
Physical biology  2012;9(6):066008.
In cellular environments, two protein molecules on their way to form a specific complex encounter many bystander macromolecules. The latter molecules, or crowders, affect both the energetics of the interaction between the test molecules and the dynamics of their relative motion. In earlier work (Zhou and Szabo 1991 J. Chem. Phys. 95 5948-52), it has been shown that, in modeling the association kinetics of the test molecules, the presence of crowders can be accounted for by their energetic and dynamic effects. The recent development of the transient-complex theory for protein association in dilute solutions makes it possible to easily incorporate the energetic and dynamic effects of crowders. The transient complex refers to a late on-pathway intermediate, in which the two protein molecules have near-native relative separation and orientation but have yet to form the many short-range specific interactions of the native complex. The transient-complex theory predicts the association rate constant as ka = ka0exp (−ΔGel*/kBT), where ka0 is the “basal” rate constant for reaching the transient complex by unbiased diffusion, and the Boltzmann factors captures the influence of long-range electrostatic interactions between the protein molecules. Crowders slow down the diffusion, therefore reducing the basal rate constant (to kac0), and induce an effective interaction energy ΔGc. We show that the latter interaction energy for atomistic proteins in the presence of spherical crowders is “long”-ranged, allowing the association rate constant under crowding to be computed as kac = kac0exp[−(ΔGel* + ΔGc*)/kBT]. Applications demonstrate that this computational method allows for realistic modeling of protein association kinetics under crowding.
PMCID: PMC3521150  PMID: 23197255
association rate constant; transient complex; macromolecular crowding; diffusion; electrostatic rate enhancement
10.  Tuning Smart Microgel Swelling and Responsive Behavior through Strong and Weak Polyelectrolyte Pair Assembly 
The layer-by-layer (LbL) assembly of polyelectrolyte pairs on temperature and pH-sensitive cross-linked poly(N-isopropylacrylamide)-co-(methacrylic acid), poly(NIPAAm-co-MAA), microgels enabled a fine tuning of the gel swelling and responsive behavior according to the mobility of the assembled polyelectrolyte (PE) pair and the composition of the outermost layer. Microbeads with well-defined morphology were initially prepared by synthesis in supercritical carbon dioxide. Upon LbL assembly of polyelectrolytes, interactions between the multilayers and the soft porous microgel led to differences in swelling and thermoresponsive behavior. For the weak PE pairs, namely poly(L-lysine) / poly(L-glutamic acid) and poly(allylamine hydrochloride) / poly(acrylic acid), polycation-terminated microgels were less swollen and more thermoresponsive than native microgel; while polyanion-terminated microgels were more swollen and not significantly responsive to temperature, in a quasi-reversible process with consecutive PE assembly. For the strong PE pair, poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate), the differences among polycation and polyanion-terminated microgels are not sustained after the first PE bilayer due to extensive ionic cross-linking between the polyelectrolytes. The tendencies across the explored systems became less noteworthy in solutions with larger ionic strength due to overall charge shielding of the polyelectrolytes and microgel. ATR FT-IR studies correlated the swelling and responsive behavior after LbL assembly on the microgels with the extent of H-bonding and alternating charge distribution within the gel. Thus, the proposed LbL strategy may be a simple and flexible way to engineer smart microgels in terms of size, surface chemistry, overall charge and permeability.
PMCID: PMC3412153  PMID: 22676290
11.  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.
PMCID: PMC3601481  PMID: 23402619
Molecular dynamics simulation; folding; villin; protein-protein interactions
12.  Tunable Encapsulation of Proteins within Charged Microgels 
Macromolecules  2011;44(20):8154-8160.
The binding of cytochrome c to pH and thermoresponsive colloidal hydrogels was investigated using multiangle light scattering, measuring loading through changes in particle molar mass and root mean square radius. Loosely cross-linked microgels [composed of a random copolymer of N-isopropylacrylamide (NIPAm) and acrylic acid (AAc)] demonstrated a high loading capacity for protein. Encapsulation was dependent on both the charge characteristics of the network and the salinity of the medium. Under favorable binding conditions (neutral pH, low ionic strength), microgels containing the highest studied charge density (30 mol% AAc) were capable of encapsulating greater than 9.7 × 105 cytochrome c molecules per particle. Binding resulted in the formation of a polymer-protein complex and condensation of the polymer. Anionic microgels demonstrated a change in density ~20-fold in the presence of oppositely charged proteins. These studies of cytochrome c encapsulation represent a significant step towards direct measurement of encapsulation efficiency in complex media as we pursue responsive nanogels and microgels for the delivery of macromolecular therapeutic agents.
PMCID: PMC3207277  PMID: 22058574
13.  Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels 
Biomaterials  2008;29(34):4481-4493.
Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with <≈100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating.
PMCID: PMC2633218  PMID: 18771802
polyethylene glycol; albumin; microgel; nanogel; surface modification; cell adhesion; protein adsorption
14.  Effects of proteins on protein diffusion 
Despite increased attention, little is known about how the crowded intracellular environment affects basic phenomena like protein diffusion. Here, we use NMR to quantify the rotational and translational diffusion of a 7.4-kDa test protein, chymotrypsin inhibitor 2 (CI2), in solutions of glycerol, synthetic polymers, proteins, and cell lysates. As expected, translational diffusion and rotational diffusion decrease with increasing viscosity. In glycerol, for example, the decrease follows the Stokes-Einstein and Stokes-Einstein-Debye laws. Synthetic polymers cause negative deviation from the Stokes Laws and affect translation more than rotation. Surprisingly, however, protein crowders have the opposite effect, causing positive deviation and reducing rotational diffusion more than translational diffusion. Indeed, bulk proteins severely attenuate the rotational diffusion of CI2 in crowded protein solutions. Similarly, CI2 diffusion in cell lysates is comparable to its diffusion in crowded protein solutions, supporting the biological relevance of the results. The rotational attenuation is independent of the size and total charge of the crowding protein, suggesting that the effect is general. The difference between the behavior of synthetic polymers and protein crowders suggests that synthetic polymers may not be suitable mimics of the intracellular environment. NMR relaxation data reveal that the source of the difference between synthetic polymers and proteins is the presence of weak interactions between the proteins and CI2. In summary, weak but non-specific, non-covalent chemical interactions between proteins appear to fundamentally impact protein diffusion in cells.
PMCID: PMC2898919  PMID: 20560582
In-cell NMR; Macromolecular crowding; Protein diffusion; Weak interactions
15.  Cell-Free Protein Expression under Macromolecular Crowding Conditions 
PLoS ONE  2011;6(12):e28707.
Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process.
Methodology/Principal Findings
Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments.
Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE.
PMCID: PMC3234285  PMID: 22174874
16.  Method to Predict Crowding Effects by Postprocessing Molecular Dynamics Trajectories: Application to the Flap Dynamics of HIV-1 Protease 
The internal dynamics of proteins inside of cells may be affected by the crowded intracellular environments. Here, we test a novel approach to simulations of crowding, in which simulations in the absence of crowders are postprocessed to predict crowding effects, against the direct approach of simulations in the presence of crowders. The effects of crowding on the flap dynamics of HIV-1 protease predicted by the postprocessing approach are found to agree well with those calculated by the direct approach. The postprocessing approach presents distinct advantages over the direct approach in terms of accuracy and speed and is expected to have broad impact on atomistic simulations of macromolecular crowding.
PMCID: PMC2837415  PMID: 20228897
17.  Network Deconstruction Reveals Network Structure in Responsive Microgels 
The journal of physical chemistry. B  2011;115(14):3761-3764.
Detailed characterization of hydrogel particle erosion revealed critical physicochemical differences between spheres, where network decomposition was informative of network structure. Real-time, in situ monitoring of the triggered erosion of colloidal hydrogels (microgels) was performed via multiangle light scattering. The solution-average molar mass and root-mean-square radii of eroding particles were measured as a function of time for microgels prepared from N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMAm), copolymerized with a chemically-labile cross-linker (1,2-dihydroxylethylene)bisacrylamide (DHEA). Precipitation polymerization was employed to yield particles of comparable dimensions but with distinct topological features. Heterogeneous cross-linker incorporation resulted in a heterogeneous network structure for pNIPAm microgels. During the erosion reaction, mass loss proceeded from the exterior towards the interior of the polymer. In contrast, pNIPMAm microgels had a more homogeneous network structure, which resulted in a more uniform mass loss throughout the particle during erosion. Although both particle types degraded into low molar mass products, pNIPAm microgels were incapable of complete dissolution due to the presence of non-degradable cross-links arising from chain transfer and branching during particle synthesis. The observations described herein provide insight into key design parameters associated with the synthesis of degradable hydrogel particles, which may be of use in various biotechnological applications.
PMCID: PMC3074247  PMID: 21425815
polymer erosion; microgel; multiangle light scattering; responsive polymer; pNIPAm; pNIPMAm
Biopolymers  2013;99(4):239-244.
The term “macromolecular crowding” denotes the combined effects of high volume fractions of nominally unrelated macromolecules upon the equilibrium and transport properties of all macrosolutes, dilute as well as concentrated, in the crowded medium. We present a formal partitioning of the total crowding effect into contributions from steric exclusion (excluded volume) and weak, nonspecific attractive interactions between a concentrated “crowding agent” and reactant and product species present at trace concentration. A numerical example of the combined effect of both steric and chemical interactions between crowder and tracer upon the reversible dimerization of tracer is presented, based upon reasonable estimates of the magnitude of both repulsive and attractive interactions between tracer and crowder species.
PMCID: PMC3557810  PMID: 23348671
Excluded volume; steric repulsion; weak binding; equivalent hard particle model
19.  Macromolecular crowding fails to fold a globular protein in cells 
Proteins perform their function in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules will stabilize globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells with a variant of protein L, a 7-kDa globular protein with seven lysine residues replaced by glutamic acids. Eighty-four percent of the variant molecules populate the denatured state in dilute buffer at room temperature, compared to 0.1% for the wild-type protein. We then used in-cell nuclear magnetic resonance spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (~1 kcal/mol) free energy deficit. The data are consistent with the idea that non-specific interactions between cytoplasmic components can overcome the excluded volume effect. Evidence for these interactions is provided by the observation that adding simple salts folds the variant in dilute solution, but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with other studies of protein stability in cells, and suggest that stabilizing excluded volume effects, which must be present under crowded conditions, can be ameliorated by non-specific interactions between cytoplasmic components.
PMCID: PMC3102155  PMID: 21534566
20.  A Generalized Fundamental Measure Theory for Atomistic Modeling of Macromolecular Crowding 
Macromolecular crowding inside cells affects the thermodynamic and kinetic properties of proteins. The scaled particle theory (SPT) has played an important role toward establishing a qualitative picture for the effects of crowding. However, SPT-based modeling lacks molecular details. Molecular dynamics simulations overcome this limitation, but at great computational cost. Here we present a theoretical method for modeling crowding at the atomic level. The method makes it possible to achieve exhaustive conformational sampling in modeling crowding effects and to tackle challenges posed by large protein oligomers and by complex mixtures of crowders.
PMCID: PMC3537823  PMID: 20365782
21.  Enhanced and sustained topical ocular delivery of cyclosporine A in thermosensitive hyaluronic acid-based in situ forming microgels 
For nearly a decade, thermoresponsive ophthalmic in situ gels have been recognized as an interesting and promising ocular topical delivery vehicle for lipophilic drugs. In this study, a series of thermosensitive copolymers, hyaluronic acid-g-poly(N-isopropylacrylamide) (HA-g-PNIPAAm), was synthesized, by coupling carboxylic end-capped PNIPAAm to aminated hyaluronic acid through amide bond linkages, and was used as a potential carrier for the topical ocular administration of cyclosporine A (CyA). The lower critical solution temperature of HA-g-PNIPAAm59 in aqueous solutions was measured as 32.7°C, which was not significantly affected by the polymer concentration. Moreover, HA-g-PNIPAAm59 microgels showed a high drug loading efficiency (73.92%) and a controlled release profile that are necessary for biomedical application. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) observations showed that HA-g-PNIPAAm microgels were spherical in shape with homogeneous size. Based on the result of the eye irritation test, the HA-g-PNIPAAm microgels formulation was shown to be safe and nonirritant for rabbit eyes. In addition, HA-g-PNIPAAm microgels achieved significantly higher CyA concentration levels in rabbit corneas (1455.8 ng/g of tissue) than both castor oil formulation and commercial CyA eye drops. Therefore, these newly described thermoresponsive HA-g-PNIPAAm microgels demonstrated attractive properties to serve as pharmaceutical delivery vehicles for a variety of ophthalmic applications.
PMCID: PMC3788692  PMID: 24092975
thermosensitive microgels; ophthalmic drug delivery; hyaluronic acid; cyclosporine A
22.  Polymeric Crowding Agents Improve Passive Biomacromolecule Encapsulation in Lipid Vesicles 
Langmuir  2010;26(16):13195-13200.
Large solutes such as high molecular weight proteins can be difficult to encapsulate in lipid vesicles. Passive trapping of these macromolecular solutes during vesicle formation typically results in concentrations inside the vesicles that are much lower than in the external solution. Here, we investigated the effect of macromolecular crowding on passive encapsulation of biological macromolecules with molecular weights ranging from 52 kDa to 660 kDa within both individual giant lipid vesicles (GVs, >3 μm diameter) and populations of 200 nm diameter large unilamellar vesicles (LUVs). Fluorescently labeled biomacromolecules were encapsulated during vesicle formation in the presence or absence of three weight percent poly(ethylene glycol) (PEG; 8 kDa) or dextran 500 kDa, which served as crowding agents. Encapsulation efficiency of the labeled biomolecules was higher for the lower molecular weight solutes, with internal concentrations essentially equal to external concentrations for labeled biomacromolecules with hydrodynamic radii (rh) less than 10 nm. In contrast, internal concentrations were reduced markedly for larger solutes with rh ≥ 10 nm. Addition of PEG or dextran during vesicle formation improved encapsulation of these larger proteins up to the same levels as observed for the smaller proteins, such that internal and external concentrations were equal. This observation is consistent with PEG and dextran acting as volume excluders, reducing the hydrodynamic radius of the biomacromolecules and increasing their encapsulation. This work demonstrates a simple and general route to improved encapsulation of otherwise poorly encapsulated macromolecular solutes in both GV and LUVs up to their concentration in the solution present during vesicle formation.
PMCID: PMC2919175  PMID: 20695558
23.  Polymer Crowders and Protein Crowders Act Similarly on Protein Folding Stability 
FEBS letters  2013;587(5):394-397.
Recently a polymer crowder and two protein crowders were found to have opposite effects on the folding stability of chymotrypsin inhibitor 2 (CI2), suggesting that they interact differently with CI2. Here we propose that all the macromolecular crowders act similarly, with an entropic component favoring the folded state and an enthalpic component favoring the unfolded state. The net effect is destabilizing below a crossover temperature but stabilizing above it. This general trend is indeed observed in recent experiments and hints experimental temperature as a reason for the opposite crowding effects of the polymer and protein crowders.
PMCID: PMC3581760  PMID: 23353683
macromolecular crowding; protein folding; crossover temperature
24.  Macromolecular Crowding and Size Effects on Probe Microviscosity 
Biophysical Journal  2008;95(11):5362-5373.
Development of biologically relevant crowding solutions necessitates improved understanding of how the relative size and density of mobile obstacles affect probe diffusion. Both the crowding density and relative size of each co-solute in a mixture will contribute to the measured microviscosity as assessed by altered translational mobility. Using multiphoton fluorescent correlation spectroscopy, this study addresses how excluded volume of dextran polymers from 10 to 500 kDa affect microviscosity quantified by measurements of calmodulin labeled with green fluorescent protein as the diffusing probe. Autocorrelation functions were fit using both a multiple-component model with maximum entropy method (MEMFCS) and an anomalous model. Anomalous diffusion was not detected, but fits of the data with the multiple-component model revealed separable modes of diffusion. When the dominant mode of diffusion from the MEMFCS analysis was used, we observed that increased excluded volume slows probe mobility as a simple exponential with crowder concentration. This behavior can be modeled with a single parameter, β, which depends on the dextran size composition. Two additional modes of diffusion were observed using MEMFCS and were interpreted as unique microviscosities. The fast mode corresponded to unhindered free diffusion as in buffer, whereas the slower agreed well with the bulk viscosity. At 10% crowder concentration, one finds a microviscosity approximately three times that of water, which mimics that reported for intracellular viscosity.
PMCID: PMC2586553  PMID: 18790853
25.  Effects of Molecular Crowding on the Dynamics of Intrinsically Disordered Proteins 
PLoS ONE  2012;7(11):e49876.
Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.
PMCID: PMC3506533  PMID: 23189168

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