Buffer effects on EcoRV kinetics as measured by fluorescent staining and digital imaging of plasmid cleavage

We have developed a protocol to quantify polymer DNA cleavage which replaces the traditional radiolabeling and scintillation counting with fluorescent staining and digital imaging. This procedure offers high sensitivity, speed, and convenience, while avoiding waste and error associated with traditional 32P radiolabeling. This protocol was used to measure cleavage of pBR322 plasmid DNA by EcoRV, a type II restriction enzyme. EcoRV was found to exhibit an order of magnitude difference in binding in two apparently similar buffers used in previous investigations. To determine the origin of this effect, we measured reaction kinetics in buffers of different chemical nature and concentration: Tris, bis-Tris propane, Tes, Hepes, and cacodylate. We found that buffer concentration and identity had significant effects on EcoRV reaction velocity through large changes in specific binding and nonspecific binding (reflected in the Michaelis constant Km and the dissociation constant for nonspecific binding Kns). There were only small changes in Vmax. The source of the buffer effect is the protonated amines common to many pH buffers. These buffer cations likely act as counterions screening DNA phosphates, where both the protonated buffer structure and concentration affect enzyme binding strength. It appears that by choosing anionic buffers or zwitterionic buffers with a buried positive charge, buffer influence on the protein binding to DNA can be largely eliminated.     

Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found a < 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.     

Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease.

The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with KAss = 4 x 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with KAss less than or equal to 1 x 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.     

Macromolecular crowding perturbs protein refolding kinetics: implications for folding inside the cell

We have studied the effects of macromolecular crowding on protein folding kinetics by studying the oxidative refolding of hen lysozyme in the absence and presence of high concentrations of bovine serum albumin and Ficoll 70. The heterogeneity characteristic of the lysozyme refolding process is preserved under crowded conditions. This, together with the observation that the refolding intermediates that accumulate to significant levels are very similar in the absence and presence of Ficoll, suggests that crowding does not alter substantially the energetics of the protein folding reaction. However, the presence of high concentrations of macromolecules results in the acceleration of the fast track of the refolding process whereas the slow track is substantially retarded. The results can be explained by preferential excluded volume stabilization of compact states relative to more unfolded states, and suggest that, relative to dilute solutions, the rates of many protein folding processes are likely to be altered under conditions that more closely resemble the intracellular environment.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.     

Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme

The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism.     

Macromolecular crowding enhances the binding of superoxide dismutase to xanthine oxidase: implications for protein-protein interactions in intracellular environments

Physiological medium constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo. Measuring protein-protein interaction in crowded solutions can mimic this environment. Here we report the application of fluorescence spectroscopy and resonant mirror biosensor to investigate the interactions of bovine milk xanthine oxidase and bovine erythrocyte copper, zinc-superoxide dismutase in crowded solutions. Four nonspecific high molecular mass crowding agents, poly(ethylene glycol) 2000 and 20,000, Ficoll 70, and dextran 70, and one low molecular mass compound, glycerol, are used. Superoxide dismutase shows a strong and macromolecular crowding agent concentration-dependent binding affinity to xanthine oxidase. Addition of high concentrations of such high molecular mass crowding agents increases the binding constant remarkably and thus stabilizes superoxide dismutase activity, compared to those in the absence of crowding agents. In contrast, glycerol has little effect on the binding constant and decreases superoxide dismutase activity over the same concentration range. Such a pattern suggests that the enhancing effects of polymers and polysaccharides on the binding are due to macromolecular crowding. Taken together, these results indicate that macromolecular crowding enhances the binding of superoxide dismutase to xanthine oxidase and is favorable to the function of superoxide dismutase.     

On the separation ability of various Ficoll gradient solutions in zonal centrifugation

Ficoll samples of various molecular weights, Mr(15-4200) X 10(3), forming gradient solutions with different separation resolutions and a more selective action have been obtained by fractionation of Ficoll 400 polysaccharide. On the basis of sedimentation-diffusion data and viscometry of Ficoll fractions in water and dimethylformamide, conclusions about the type of branching of macromolecules and the intensity of intramolecular hydrodynamic interaction were made; the size and shape asymmetry of Ficoll molecules were calculated in connection with their mobility in solution.     

Soft interactions and volume exclusion by polymeric crowders can stabilize or destabilize transient structure in disordered proteins depending on polymer concentration

The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well‐understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L^?1 and 400 g L^?1 of Ficoll, a synthetic polysaccharide, using a recently‐developed strong cation exchange‐based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275–1282]. Transiently helical regions of ACTR exchanged faster in 300 g L^?1 Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L^?1 Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L^?1 Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L^?1 Ficoll is a semi‐dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468–1479. © 2017 Wiley Periodicals, Inc.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.     

Investigation of the inhibitory role of phosphorothioate internucleotidic linkages on the catalytic activity of the restriction endonuclease EcoRV

The inhibitory effect of phosphorothioate residues, located within one strand of double-stranded DNA, on the hydrolytic activity of the restriction endonuclease EcoRV was investigated. Specific incorporation of a phosphorothioate group at the site of cleavage yielded the sequence 5'-GATsATC-3'. This modified sequence was cleaved at a relative rate of 0.1 compared to the unmodified substrate. Substrates 5'-GATsAsTC-3' and 5'-GsATsATC-3', both containing one additional phosphorothioate substitution, were linearized at a rate of 0.04 relative to unmodified DNA. However, under the same conditions, fully dAMPS-substituted DNA was found to be virtually resistant to the hydrolytic activity of EcoRV. Further experiments showed that double-stranded DNA fragments generated by PCR containing phosphorothioate groups within both strands are potent inhibitors of EcoRV catalysis. The inhibition was independent of whether the inhibitor fragment contained an EcoRV recognition site. We concluded that substitution of the phosphate group at the site of cleavage by a phosphorothioate residue decreases the rate of EcoRV-catalyzed hydrolysis most significantly. Substitution of other phosphate groups within the recognition sequence plays a limited role in enzyme inhibition. The presence of multiple dNMPS residues at regions of the DNA removed from the EcoRV recognition site may decrease the amount of enzyme available for catalysis by nonspecific binding to EcoRV.     

Reactions of BglI and other type II restriction endonucleases with discontinuous recognition sites

Type II restriction enzymes generally recognize continuous sequences of 4-8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA. BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV. BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism.     

Excluded volume as a determinant of macromolecular structure and reactivity

The effect of excluded volume on the thermodynamic activity of globular macromolecules and macromolecular complexes in solution is studied in the hard-particle approximation. Activity coefficients are calculated as a function of the fraction of total volume occupied by macromolecules using relations obtained from scaled particle and lattice models. Significant and readily observable effects are predicted to occur as the fraction of volume occupied by globular macromolecules increases, including the following: (i) Compact quasi-spherical macromolecular conformations become increasingly energetically favored over extended anisometric conformations. (ii) Self- and heteroassociation processes are enhanced, particularly those leading to the formation of compact quasi-spherical aggregates. (iii) Depending upon the details of the reaction mechanism, the rate of an enzyme-catalyzed reaction may monotonically decrease, go through a maximum, or exhibit more complex behavior. A given degree of volume occupancy by larger macromolecules is predicted to have less effect on the structure and self-association of smaller macromolecules than the same degree of volume occupancy by smaller macromolecules has on the structure and self-association of larger macromolecules.     

Kinetic analyses of divalent cation-dependent EcoRV digestions on a DNA-immobilized quartz crystal microbalance

Enzymatic digestion with a type IIP restriction endonuclease EcoRV was investigated on a DNA-immobilized 27-MHz quartz crystal microbalance (QCM). Real-time observations of both the enzyme binding process and the DNA cleavage process of EcoRV were followed by frequency (mass) changes on the QCM, which were dependent on divalent cations such as Ca(2+) or Mg(2+). In the presence of Ca(2+), the site-specific binding of EcoRV to DNA could be observed, without the catalytic process. On the other hand, in the presence of Mg(2+), both the binding of the enzyme to the specific DNA (mass increase) and the site-specific cleavage reaction (mass decrease) could be observed continuously from QCM frequency changes. From time courses of frequency (mass) changes, each kinetic parameter, namely binding rate constants (k(on)), dissociation rate constants (k(off)), dissociation constants (K(d)) of EcoRV to DNA, and catalytic rate constant (k(cat)) of the cleavage reaction, could be determined. The binding kinetic parameters of EcoRV in the presence of Ca(2+) were consistent with those of the binding process followed by the cleavage process in the presence of Mg(2+). The k(cat) value obtained by the QCM method was also consistent with that obtained by other methods. This study is the first to simultaneously determine k(on), k(off), and k(cat) for a type IIP restriction endonuclease on one device.     

Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease.

Measurements of binding equilibria of EcoRV endonuclease to DNA, for a series of base-analogue substrates, demonstrate that expression of sequence selectivity is strongly enhanced by the presence of Ca2+ ions. Binding constants were determined for short duplex oligodeoxynucleotides containing the cognate DNA site, three cleavable noncognate sites, and a fully nonspecific site. At pH 7.5 and 100 mM NaCl, the full range of specificity from the specific (tightest binding) to nonspecific (weakest binding) sites is 0.9 kcal/mol in the absence of metal ions and 5.8 kcal/mol in the presence of Ca2+. Precise determination of binding affinities in the presence of the active Mg2+ cofactor was found to be possible for substrates retaining up to 1.6% of wild-type activity, as determined by the rate of phosphoryl transfer. These measurements show that Ca2+ is a near-perfect analogue for Mg2+ in binding reactions of the wild-type enzyme with DNA base-analogue substrates, as it provides identical DeltaDeltaG degrees bind values among the cleavable noncognate sites. Equilibrium dissociation constants of wild-type and base-analogue sites were also measured for the weakly active EcoRV mutant K38A, in the presence of either Mg2+ or Ca2+. In this case, Ca2+ allows expression of a greater degree of specificity than does Mg2+. DeltaDeltaG degrees bind values of K38A toward specific versus nonspecific sites are 6.1 kcal/mol with Ca2+ and 3.9 kcal/mol with Mg2+, perhaps reflecting metal-specific conformational changes in the ground-state ternary complexes. The enhancement of binding specificity provided by divalent metal ions is likely to be general to many restriction endonucleases and other metal-dependent nucleic acid-modifying enzymes. These results strongly suggest that measurements of DNA binding affinities for EcoRV, and likely for many other restriction endonucleases, should be performed in the presence of divalent metal ions.     

Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo

The EcoRV restriction/modification system consists of two enzymes that recognize the DNA sequence GATATC. The EcoRV restriction endonuclease cleaves DNA at this site, but the DNA of Escherichia coli carrying the EcoRV system is protected from this reaction by the EcoRV methyltransferase. However, in vitro, the EcoRV nuclease also cleaves DNA at most sites that differ from the recognition sequence by one base pair. Though the reaction of the nuclease at these sites is much slower than that at the cognate site, it still appears to be fast enough to cleave the chromosome of the cell into many fragments. The possibility that the EcoRV methyltransferase also protects the noncognate sites on the chromosome was examined. The modification enzyme methylated alternate sites in vivo, but these were not the same as the alternate sites for the nuclease. The excess methylation was found at GATC sequences, which are also the targets for the dam methyltransferase of E. coli, a protein that is homologous to the EcoRV methyltransferase. Methylation at these sites gave virtually no protection against the EcoRV nuclease: even when the EcoRV methyltransferase had been overproduced, the cellular DNA remained sensitive to the EcoRV nuclease at its noncognate sites. The viability of E. coli carrying the EcoRV restriction/modification system was found instead to depend on the activity of DNA ligase. Ligase appears to proofread the EcoRV R/M system in vivo: DNA, cut initially in one strand at a noncognate site for the nuclease, is presumably repaired by ligase before the scission of the second strand.     

Binding and recognition of GATATC target sequences by the EcoRV restriction endonuclease: a study using fluorescent oligonucleotides and fluorescence polarization

Oligonucleotides labeled with hexachlorofluorescein (hex) have enabled the interaction of the restriction endonuclease EcoRV with DNA to be evaluated using fluorescence anisotropy. The sensitivity of hex allowed measurements at oligonucleotide concentrations as low as 1 nM, enabling K(D) values in the low nanomolar range to be measured. Both direct titration, i.e., addition of increasing amounts of the endonuclease to hex-labeled oligonucleotides, and displacement titration, i.e., addition of unlabeled oligonucleotide to preformed hex-oligonucleotide/EcoRV endonuclease complexes, have been used for K(D) determination. Displacement titration is the method of choice; artifacts due to any direct interaction of the enzyme with the dye are eliminated, and higher fluorescent-labeled oligonucleotide concentrations may be used, improving signal-to-noise ratio. Using this approach (with three different oligonucleotides) we found that the EcoRV restriction endonuclease showed a preference of between 1.5 and 6.5 for its GATATC target sequence at pH 7.5 and 100 mM NaCl, when the divalent cation Ca2+ is absent. As expected, both the presence of Ca2+ and a decrease in pH value stimulated the binding of specific sequences but had much less effect on nonspecific ones.     

EcoRV restriction endonuclease binds all DNA sequences with equal affinity

In the presence of MgCl2, the EcoRV restriction endonuclease cleaves its recognition sequence on DNA at least a million times more readily than any other sequence. In this study, the binding of the EcoRV restriction enzyme to DNA was examined in the absence of Mg2+. With each DNA fragment tested, several DNA-protein complexes were detected by electrophoresis through polyacrylamide. No differences were observed between isogenic DNA molecules that either contained or lacked the EcoRV recognition site. The number of complexes with each fragment varied with the length of the DNA. Three complexes were formed with a DNA molecule of 55 base pairs, corresponding to the DNA bound to 1, 2, or 3 molecules of the protein, while greater than 15 complexes were formed with a DNA of 381 base pairs. A new method was developed to analyze the binding of a protein to multiple sites on DNA. The method showed that the EcoRV enzyme binds to all DNA sequences, including the EcoRV recognition site, with the same equilibrium constant, though two molecules of the protein bind preferentially to adjacent sites on the DNA in a cooperative fashion. All of the complexes with a substrate that contained the EcoRV site dissociated upon addition of competitor DNA, but when the competitor was mixed with MgCl2, a fraction of the substrate was cleaved at the EcoRV site. The fraction cleaved was due mainly to the translocation of the enzyme from nonspecific sites on the DNA to the specific site.     

Tracer diffusion in F-actin and Ficoll mixtures. Toward a model for cytoplasm.

We have previously reported that self-diffusion of inert tracer particles in the cytoplasm of living Swiss 3T3 cells is hindered in a size-dependent manner (Luby-Phelps, K., D.L. Taylor, and F. Lanni. 1986. J. Cell Biol. 102:2015-2022; Luby-Phelps, K., P.E. Castle, D.L. Taylor, and F. Lanni. 1987. Proc Natl. Acad. Sci. USA. 84:4910-4913). Lacking a theory that completely explains our data, we are attempting to understand the molecular architecture responsible for this phenomenon by studying tracer diffusion in simple, reconstituted model systems. This report contains our findings on tracer diffusion in concentrated solutions of Ficoll 70 or Ficoll 400, in solutions of entangled F-actin filaments, and in solutions of entangled F-actin containing a background of concentrated Ficoll particles or concentrated bovine serum albumin (BSA). A series of size-fractionated fluorescein-Ficolls were used as tracer particles. By fluorescence recovery after photobleaching (FRAP), we obtained the mean diffusion coefficients in a dilute, aqueous reference phase (Do), the mean diffusion coefficients in the model matrices (D), and the mean hydrodynamic radii (RH) for selected tracer fractions. For each model matrix, the results were compared with similar data obtained from living cells. As in concentrated solutions of globular proteins (Luby-Phelps et al., 1987), D/Do was not significantly size-dependent in concentrated solutions of Ficoll 400 or Ficoll 70. In contrast, D/Do decreased monotonically with increasing RH in solutions of F-actin ranging in concentration from 1 to 12 mg/ml. This size dependence was most pronounced at higher F-actin concentrations. However, the shape of the curve and the extrapolated value of D/Do in the limit, RH----O did not closely resemble the cellular data for tracers in the same size range (3 less than RH less than 30 nm). In mixtures of F-actin and Ficoll or F-actin and BSA, D/Do was well approximated by D/Do for the same concentration of F-actin alone multiplied by D/Do for the same concentrations of Ficoll or BSA alone. Based on these results, it is possible to model the submicroscopic architecture of cytoplasm in living cells as a densely entangled filament network (perhaps made up of F-actin and other filamentous structures) interpenetrated by a fluid phase crowded with globular macromolecules, which in cytoplasm would be primarily proteins.     

Competitive Interactions of Ligands and Macromolecular Crowders with Maltose Binding Protein

Cellular signaling involves a cascade of recognition events occurring in a complex environment with high concentrations of proteins, polysaccharides, and other macromolecules. The influence of macromolecular crowders on protein binding affinity through hard-core repulsion is well studied, and possible contributions of protein-crowder soft attraction have been implicated recently. Here we present direct evidence for weak association of maltose binding protein (MBP) with a polysaccharide crowder Ficoll, and that this association effectively competes with the binding of the natural ligand, maltose. Titration data over wide ranges of maltose and Ficoll concentrations fit well with a three-state competitive binding model. Broadening of MBP ¹H­¹⁵N TROSY spectra by the addition of Ficoll indicates weak protein-crowder association, and subsequent recovery of sharp NMR peaks upon addition of maltose indicates that the interactions of the crowder and the ligand with MBP are competitive. We hypothesize that, in the Escherichia coli periplasm, the competitive interactions of polysaccharides and maltose with MBP could allow MBP to shuttle between the peptidoglycan attached to the outer membrane and the ATP-binding cassette transporter in the inner membrane.