Effect of crowding by dextrans and Ficolls on the rate of alkaline phosphatase-catalyzed hydrolysis: a size-dependent investigation

The cell cytosol is crowded with macromolecules such as proteins, nucleic acids, and membranes. The consequences of such crowding remain unclear. How is the rate of a typical enzymatic reaction, involving a freely diffusing enzyme and substrate, affected by the presence of macromolecules of different sizes, shapes, and concentrations? Here, we mimic the cytosolic crowding in vitro, using dextrans and Ficolls, for the first time in a variety of sizes ranging from 15 to 500 kDa, in a concentration range 0–30% w/w. Alkaline phosphatase–catalyzed hydrolysis of p‐nitrophenyl phosphate (PNPP) was chosen as the model reaction. A pronounced decrease in the rate with increase in fractional volume occupancy of dextran is observed for larger dextrans (200 and 500 kDa) in contrast to smaller dextrans (15–70 kDa). Our results indicate that, at 20% w/w, smaller dextrans (15–70 kDa) reduce the initial rate moderately (1.4‐ to 2.4‐fold slowing), while larger dextrans (>200 kDa) slow the reaction considerably (>5‐fold). Ficolls (70 and 400 kDa) slow the reaction moderately (1.3‐ to 2.3‐fold). The influence of smaller dextrans was accounted by a combination of increase in viscosity as sensed by PNPP and a minor offsetting increase in enzyme activity due to crowding. Larger dextrans apparently reduce the frequency of enzyme substrate encounter. The reduced influence of Ficolls is attributed to their compact and quasispherical shape, much unlike the dextrans. © 2006 Wiley Periodicals, Inc. Biopolymers 83: 477–486, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Interaction of dextran sulfate with phospholipid surfaces and liposome aggregation and fusion

The binding of dextran sulfate to phospholipid liposomes was investigated by microelectrophoresis experiments. The polyanion binds to neutral phospholipid liposomes (DMPC and PE) only in the presence of Ca2+. If positively charged stearylamine is incorporated in the vesicles dextran sulfate is bound without Ca2+. Negatively charged phospholipids as PS do not bind dextran sulfate, even in the presence of millimolar concentrations of Ca2+. The adsorption of dextran sulfate results in an aggregation of vesicles due to a bridging mechanism. In all cases the aggregation is followed by a disaggregation toward higher dextran sulfate concentrations. The disaggregation process starts at polymer concentrations smaller than the concentration of the onset of saturation of the adsorption. By use of the probe dilution method a fusion of small DMPC and DMPC/PE vesicles in the presence of Ca2+ and dextran sulfate was found.     

Thermodynamics of substrate binding to the chaperone SecB

The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and alpha-lactalbumin. SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded disulfide intermediates of alpha-lactalbumin, which have 40-60% of native secondary structure. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29, and -0.41 kcal mol(-1) K(-1), respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. There is no evidence for two separate types of binding sites for positively charged and hydrophobic ligands. Spectroscopic and proteolysis protection studies of the binding of SecB to poly-L-Lys show that binding of highly positively charged peptide ligands to negatively charged SecB leads to charge neutralization and subsequent aggregation of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft. SecB aggregation in the absence of substrate is prevented by electrostatic repulsion between negatively charged SecB tetramers.     

Electrostatic interaction in the NH2-terminus accelerates inactivation of the Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signalling properties of nerve and muscle cells. While it has been assumed that the rapid inactivation of the Kv1.4 channel is controlled by a “ball and chain” inactivation mechanism, the chain structure of the channel has not been well defined. Here, by conducting electrophysiological studies on variants containing mutations of the positively charged and negatively charged segments of the NH₂-terminal of the channel protein, we show that neutralization or deletion of the positively charged segment (residues 83-98) significantly slowed the inactivation process. Replacement of this positively charged segment with the negatively charged segment (residues 123-137), and vice versa, so that both segments were simultaneously positively or negatively charged, also slowed the inactivation process. Furthermore, the inactivation process was not changed when the positively charged and the negatively charged segments were interchanged. In contrast, the voltage dependence of activation and inactivation of the channels was not significantly altered by these mutants. These results indicate that the electrostatic interaction between the positively and negatively charged segments plays a critical role in the inactivation process of the Kv1.4 channel. Taken together, we propose that the electrostatic interaction accelerates the inactivation of the Kv1.4 channel by making it easier for the inactivation ball to access its binding site.     

Adsorption of RNase A on cationic polyelectrolyte brushes: a study by isothermal titration calorimetry

We present a study of the adsorption of a positively charged protein to a positively charged spherical polyelectrolyte brush (SPB) by isothermal titration calorimetry (ITC). ITC is used to determine the adsorption isotherm as a function of temperature and of salt concentration (at physiological pH 7.2). At low ionic strength, RNase A is strongly adsorbed by the SPB particles despite the fact that both the SPB particles and the protein are positively charged. Virtually no adsorption takes place when the ionic strength is raised through added salt. This is strong evidence for counterion release as the primary driving force for protein adsorption. We calculated that ~2 counterions were released upon RNase A binding. The adsorption of RNase A into like-charged SPB particles is entropy-driven, and protein protonation was not significant. Temperature-dependent measurements showed a disagreement between the enthalpy derived via the van't Hoff equation and the calorimetric enthalpy. Further analysis shows that van't Hoff analysis leads to the correct enthalpy of adsorption. The additional contributions to the measured enthalpy are potentially sourced from unlinked equilibria such as conformational changes that do not contribute to the binding equilibrium.     

Initial steps in protein membrane insertion. Bacteriophage M13 procoat protein binds to the membrane surface by electrostatic interaction.

Bacteriophage M13 procoat protein is synthesized on free polysomes prior to its assembly into the inner membrane of Escherichia coli. As an initial step of the membrane insertion pathway, the precursor protein interacts with the cytoplasmic face of the inner membrane. We have used oligonucleotide-directed mutagenesis to study the regions of the procoat protein involved in membrane binding. We find that there is an absolute requirement for positively charged amino acids at both ends of the protein. Replacing these with negatively charged residues resulted in an accumulation of the precursor in the cytoplasm. We propose that the positively charged amino acids are directly involved in membrane binding, possibly directly to the negatively charged phospholipid head groups. This was tested in vitro with artificial liposomes. Whereas wild-type procoat interacted with these liposomes, we found that procoat mutants with negatively charged amino acids at both ends did not bind. Therefore, we conclude that newly synthesized M13 procoat protein binds electrostatically to the negatively charged inner membrane of E. coli.     

Towards biocompatible vaccine delivery systems: interactions of colloidal PECs based on polysaccharides with HIV-1 p24 antigen

This work reports on the interactions of a model protein (p24, the capside protein of HIV-1 virus) with colloids obtained from polyelectrolyte complexes (PECs) involving two polysaccharides: chitosan and dextran sulfate (DS). The PECs were elaborated by a one-shot addition of default amounts of one counterpart to the polymer in excess. Depending on the nature of the excess polyelectrolyte, the submicrometric colloid was either positively or negatively charged. HIV-1 capsid p24 protein was chosen as antigen, the ultrapure form, lipopolysaccharide-free (endotoxin-, vaccine grade) was used in most experiments, as the level of purity of the protein had a great impact on the immobilization process. p24 sorption kinetics, isotherms, and loading capacities were investigated for positively and negatively charged particles of chitosans and dextran sulfates differing in degrees of polymerization (DP) or acetylation (DA). Compared with the positive particles, negatively charged colloids had higher binding capacities, faster kinetics, and a better stability of the adsorbed p24. Capacities up to 600 mg x g(-1) (protein-colloid) were obtained, suggesting that the protein interacted within the shell of the particles. Small-angle X-rays scattering experiments confirmed this hypothesis. Finally, the immunogenicity of the p24-covered particles was assessed for vaccine purposes in mice. The antibody titers obtained with immobilized p24 was dose dependent and in the same range as for Freund's adjuvant, a gold standard for humoral responses.     

Quantitative intravital microscopy using a Generalized Polarity concept for kidney studies

In this article, we describe a ratiometric intravital two-photon microscopy technique for studying glomerular permeability and differences in proximal tubule cell reabsorption. This quantitative approach is based on the Generalized Polarity (GP) concept, in which the intensity difference between two fluorescent molecules is normalized to the total intensity produced by the two dyes. After an initial intravenous injection of a mixture of 3-, 40-, and 70-kDa fluorescently labeled dextrans into live Munich-Wistar-Frömter (MWF) rats, we were able to monitor changes in the GP values between any two dyes within local regions of the kidney, including the glomerulus, Bowman's capsule, proximal tubule lumens and proximal tubule cells, and individual capillary vessels. We were able to quantify accumulations of different dextrans in the Bowman's space and in tubular lumens as well as reabsorption by proximal tubular cells at different time points in the same rat. We found that for 6- to 8-wk-old MWF rats that developed spontaneous albuminuria, the 40- and 70-kDa dextrans, with hydrodynamic radii larger than albumin, were differentially filtered, but both were able to pass the glomerular filtration barrier and enter into the urinary space of the Bowman's capsule within a few seconds after intravenous infusion. Using GP image analysis, we found that negatively charged dextrans of both 40 and 70 kDa were better reabsorbed by the proximal tubule cells than the neutrally charged 40-kDa dextran. These results demonstrate the potential power of the GP imaging technique for quantitative studies of glomerular filtration and tubular reabsorption. glomerular permeability; tubular reabsorption; charge selectivity; two-photon excitation; multiphoton     

Interaction of glucosyltransferase from Streptococcus mutans with various glucans

Cell-free glucosyltransferase of Streptococcus mutans strain B13 (serotype d) exclusively synthesized water-insoluble glucan from sucrose. The insoluble glucan possessed strong glucan-associated glucosyltransferase activity even after extensive washing and lyophilization. Furthermore, cell-free glucosyltransferase became bound to heat-treated water-insoluble glucan or to heat-treated S. mutans B13 cells grown in Todd Hewitt broth, and the resulting glucan and cells adhered to a glass surface in the presence of exogenous sucrose. No other water-insoluble glucans bound significant quantities of glucosyltransferase. Glucan synthesis by free or glucan-bound glucosyltransferase was stimulated by low concentrations (1 to 5 mg ml-1) of isomaltose or water-soluble dextrans of various molecular weights, but higher concentrations (10 mg ml-1) inhibited glucan synthesis. The glucan synthesized in the presence of primer dextrans exhibited a reduced ability to adhere to a glass surface. Certain sugars such as maltose and fructose significantly lowered the yield of insoluble glucans. Preincubation of glucosyltransferase with the low molecular weight dextran T10 increased subsequent binding to S. mutans B13 insoluble glucan, whereas preincubation with higher molecular weight dextrans significantly inhibited the glucosyltransferase binding.     

Evaluating the Effect of Ionic Strength on Duplex Stability for PNA Having Negatively or Positively Charged Side Chains

The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the uncharged PNA backbone and negatively charged DNA or RNA backbone. However, there are no previously reported studies that systematically evaluate the effect of ionic strength on duplex stability for PNA having a charged backbone. Here we investigate the role of charge repulsion in PNA binding by synthesizing PNA strands having negatively or positively charged side chains, then measuring their duplex stability with DNA or RNA at varying salt concentrations. At low salt concentrations, positively charged PNA binds more strongly to DNA and RNA than does negatively charged PNA. However, at medium to high salt concentrations, this trend is reversed, and negatively charged PNA shows higher affinity for DNA and RNA than does positively charged PNA. These results show that charge screening by counterions in solution enables negatively charged side chains to be incorporated into the PNA backbone without reducing duplex stability with DNA and RNA. This research provides new insight into the role of electrostatics in PNA binding, and demonstrates that introduction of negatively charged side chains is not significantly detrimental to PNA binding affinity at physiological ionic strength. The ability to incorporate negative charge without sacrificing binding affinity is anticipated to enable the development of PNA therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA.     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: a new fluorescence displacement titration technique for characterizing the nucleotide binding site on the catalytic subunit

We determined the dissociation constant (Kd) of a series of nucleotides for the bovine skeletal muscle type II catalytic subunit by displacing lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) with increasing concentrations of competing nucleotide. The Kd of each nucleotide was calculated from the decreases in the fluorescence polarization of lin-benzo-ADP that accompany its displacement from the catalytic subunit. We found that modifications of the adenine moiety reduce nucleotide affinity for the enzyme. The effect was most pronounced with modifications at position 6 of the base. Replacement of the 3'-hydroxyl group of ribose with a hydrogen increased the affinity of the nucleotide; addition of phosphate to the 2'- or 3'-hydroxyl groups, on the other hand, decreased nucleotide affinity. MgATP and MgADP exhibited Kd's of about 10 microM. AMP, which contains a negatively charged alpha-phosphate, bound with reduced affinity (643 microM). Adenosine, which lacks a charged alpha-phosphate, bound with a higher affinity (32 microM). To learn more about the nature of the alpha-phosphate binding site, a series of uncharged and positively charged derivatives of the 5'-position on the ribose moiety was prepared. The uncharged derivatives bound with much greater affinity than the negatively charged AMP. The Kd's for 5'-tosyladenosine and 5'-iodo-5'-deoxyadenosine were 30 and 32 microM, respectively. Like the negatively charged AMP, positively charged derivatives also bound less tenaciously than the neutral species. The positively charged 5'-amino-5'deoxyadenosine, for example, exhibited a 15-fold higher Kd (506 microM) than the neutral congenors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ion-exchange resins greatly facilitate refolding of like-charged proteins at high concentrations

Protein refolding is a crucial step for the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high‐efficiency protein refolding. We have herein found that electrostatic repulsion between like‐charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding is extensively demonstrated with three different proteins and four kinds of ion‐exchange resins when the protein and ion‐exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect is significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. It was not realistic by any other approaches. The working mechanism of the like‐charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating protein folding. The refolding method with like‐charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. All the characters are desirable for preparative refolding of therapeutic proteins expressed in bacteria as inclusion bodies. Bioeng. 2011; 108:1068–1077. © 2010 Wiley Periodicals, Inc.     

Low-density-lipoprotein binding by mast-cell granules. Demonstration of binding of apolipoprotein B to heparin proteoglycan of exocytosed granules.

To study the interaction between low-density lipoprotein (LDL) and granules from rat serosal mast cells in vitro, mast cells were stimulated with the degranulating agent 48/80 to induce exocytosis of the secretory granules. Subsequent incubation of the exocytosed granules with 125I-LDL resulted in binding of the labelled LDL to the granules. When increasing amounts of agent 48/80 were added to mast-cell suspensions, a dose-dependent release of granules was observed and a parallel increase in the amount of 125I-LDL bound to granules resulted. 125I-LDL bound to a single class of high-affinity binding sites on the granules. At saturation, 105 ng of LDL were bound per microgram of granule protein. The lipoprotein binding to mast-cell granules was apolipoprotein(apo)-B + E-specific. Thus 125I-LDL binding to the granules was effectively compared for by LDL (apo-B) or by dimyristoyl phosphatidylcholine vesicles containing apo-E, but not by high-density lipoprotein (HDL3) containing apo-AI as their major protein component. Neutralization by acetylation of the positively charged amino groups of apo-B of LDL or presence of a high ionic strength in the incubation medium prevented LDL from binding to the granules, indicating the presence of ionic interactions between the positively charged amino acids of LDL and negatively charged groups of the granules. It could be demonstrated that LDL bound to the negatively charged heparin proteoglycan of the granules. Thus treatment of granules with heparinase resulted in loss of their ability to bind LDL, and substances known to bind to heparin, such as Toluidine Blue, avidin, lipoprotein lipase, fibronectin and protamine, all effectively competed with LDL for binding to the granules. The results show that LDL is efficiently bound to the heparin proteoglycan component of mast-cell granules once the mast cells are stimulated to release their granules into the extracellular space.     

Effect of water-soluble polymers on the state of aggregation, vesicle size, and phase transformations in mixtures of phosphatidylcholine and sodium cholate.

The state of aggregation and the steady-state size of mixed aggregates made of phospholipids and surfactants are both determined by the surfactant/lipid ratio in the mixed aggregates (Re). Water-soluble polymers, such as dextrans and polyethylene glycols (PEGs) of different molecular weights, induce reversible aggregation of phospholipid vesicles, mostly due to dehydration of the vesicle surface and depletion forces, and only at much higher concentrations, PEGs (but not dextran) also induce irreversible size growth of the vesicles. Here we show that the water-soluble polymers dextrans and PEGs do not affect the vesicle-micelle phase boundaries in mixtures of phosphatidylcholine and the anionic surfactant sodium cholate. By contrast, these polymers affect markedly the steady-state size of cholate-containing vesicles. As compared with pure phosphatidylcholine vesicles, the cholate-containing vesicles have a lower tendency to undergo polymer-induced aggregation, probably due to the electrostatic repulsion between the negatively charged vesicles, but a higher tendency to undergo irreversible size growth at relatively low polymer concentrations. Such irreversible size growth was observed not only for PEG but also for dextran, which in the absence of cholate is incapable of inducing vesicle size growth. These findings are consistent with the prevailing concept that the polymer-induced size growth is due to the effect of large structural fluctuations in the bilayers of deformed aggregated vesicles, the surface of which is dehydrated by the polymer. The presence of cholate in the bilayers at sufficiently high concentrations induces such fluctuations, yielding irreversible size growth within the clusters of dehydrated vesicles formed upon mixing with polymers.     

Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments. Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth [Abrams, F. S., and London, E. (1992) Biochemistry 31, 5312-5322]. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains. The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain. Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface. In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding. These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical. In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes. These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound. Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions.     

Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding

Phosphorylation is an important post‐translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high‐resolution protein structures from the Protein Data Bank including their co‐crystallized non‐covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature‐reported selected events. Proteins 2016; 84:565–579. © 2016 Wiley Periodicals, Inc.     

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.     

Photobleaching of the photoactive yellow protein from Ectothiorhodospira halophila promotes binding to lipid bilayers: evidence from surface plasmon resonance spectroscopy.

The photoactive yellow protein (PYP) from the phototrophic bacterium Ectothiorhodospira halophila is a small, soluble protein that undergoes reversible photobleaching upon blue light irradiation and may function to mediate the negative phototactic response. Based on previous studies of the effects of solvent viscosity and of aliphatic alcohols on PYP photokinetics, we proposed that photobleaching is concomitant with a protein conformational change that exposes a hydrophobic region on the protein surface. In the present investigation, we have used surface plasmon resonance (SPR) spectroscopy to characterize the binding of PYP to lipid bilayers deposited on a thin silver film. SPR spectra demonstrate that the net negatively charged PYP molecule can bind in a saturable manner to electrically neutral, net positively, and net negatively charged bilayers. Illumination with either blue or white light of a PYP solution, which is in contact with the bilayer, at concentrations below saturation results in an increase in the extent of binding, consistent with exposure of a high affinity hydrophobic surface in the photobleached state, a property that may contribute to its biological function. A value for the thickness of the bound PYP layer (23 A), obtained from theoretical fits to the SPR spectra, is consistent with the structure of the protein determined by x-ray crystallography and indicates that the molecule binds with its long axis parallel to the membrane surface.     

Blocking of the receptor-mediated invasion of erythrocytes by Plasmodium knowlesi malaria with sulfated polysaccharides and glycosaminoglycans

Invasion of human erythrocytes by Plasmodium knowlesi requires the Duffy blood group antigen. P. knowlesi merozoites synthesize a 135-kDa polypeptide which binds to the Duffy antigen with receptor-like specificity. In this study, we show that the sulfated polysaccharide fucoidan and the glycosaminoglycan dextran sulfate inhibit the binding of the 135-kDa polypeptide to human Duffy-positive and rhesus erythrocytes while the chondroitin sulfates do not. Fucoidan and dextran sulphate also blocked the in vitro invasion of human Duffy b and rhesus erythrocytes cells by P. knowlesi merozoites. These inhibitors were more effective at blocking the binding of the 135-kDa polypeptide to human Duffy b erythrocytes than to rhesus erythrocytes, which correlated with them having a greater inhibitory effect on invasion of merozoites into human than into rhesus erythrocytes. The blocking by these sulfated sugars is not related to charge density on the polysaccharides; fucoidan with a relatively low charge density blocks binding of the 135-kDa polypeptide at 4 micrograms/ml, while the highly negatively charged chondroitin sulfates do not block binding even at the concentration of 1 mg/ml. Furthermore, fucoidan-Sepharose bound and removed the 135-kDa polypeptide from parasite culture supernatants with a selectivity equal to that of the Duffy blood group antigen. The negatively charged sulfate groups on fucoidan and dextran sulfate and the conformation in which they are held possibly mimic similarly charged groups on the Duffy antigen which bind the 135-kDa P. knowlesi polypeptide.     

High‐throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high‐throughput small‐scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two‐step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two‐step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:516–520, 2014