Reversible polymers formed from self-complementary monomers using quadruple hydrogen bonding

Units of 2-ureido-4-pyrimidone that dimerize strongly in a self-complementary array of four cooperative hydrogen bonds were used as the associating end group in reversible self-assembling polymer systems. The unidirectional design of the binding sites prevents uncontrolled multidirectional association or gelation. Linear polymers and reversible networks were formed from monomers with two and three binding sites, respectively. The thermal and environmental control over lifetime and bond strength makes many properties, such as viscosity, chain length, and composition, tunable in a way not accessible to traditional polymers. Hence, polymer networks with thermodynamically controlled architectures can be formed, for use in, for example, coatings and hot melts, where a reversible, strongly temperature-dependent rheology is highly advantageous.     

The effect of soy isoflavones on the development of intestinal neoplasia in ApcMin mouse

Large polymeric 3-alkylpyridinium salts have been isolated from the marine sponge Reniera sarai. They are composed of N-butyl(3-butylpyridinium) repeating subunits, polymerized head-to-tail, and exist as a mixture of two main polymers with molecular weights without counterion of about 5520 and 18900. The monomer analogue of the inhibitor, N-butyl-3-butylpyridinium iodide has been synthesized. This molecule shows mixed reversible inhibition of acetylcholinesterase. The polymers also act as acetylcholinesterase inhibitors and show an unusual inhibition pattern. We tentatively describe it as quick initial reversible binding, followed by slow binding or irreversible inhibition of the enzyme. This kinetics suggests that there are several affinity binding sites on the acetylcholinesterase molecule where the polymer can bind. The first binding favors binding to other sites which leads to an apparently irreversibly linked enzyme-inhibitor complex.     

The effect of gentamicin on acetylsalicylic acid-induced platelet antiaggregatory action in mouse pial arterioles

Proton nuclear magnetic resonance (1H-NMR) spectroscopy is used to identify a preferred binding site for uncharged hydrophilic polymers on the surface of hen egg-white lysozyme. Chemical shift titrations show that exchangeable proton signals from amino acids Arg-61, Trp-62, Trp-63, Arg-73, Lys-96 and Asp-101 are selectively perturbed upon binding of poly(ethylene oxide), poly(ethylene glycol) and poly(ethylene-co-propylene oxide). The greatest binding-induced chemical shift changes are observed for Trp-62, Arg-61 and Arg-73 at the edge of the active site cleft of the protein, consistent with a predominantly hydrophobic interaction mode involving the polymer ethylene moieties. The more hydrophilic species poly(dihydroxypropyl methacrylate) causes similar but substantially smaller chemical shift effects than the other polymers, confirming the nature of the interaction. A dissociation constant of 76+/-5 mM is determined for the poly(ethylene glycol)-lysozyme complex. The relatively low affinity of the protein-polymer interactions compared to oligosaccharide substrate binding suggests that lysozyme activity is minimally affected by these materials.     

Affinity precipitation of proteins

Affinity precipitation is being studied as a technique to be introduced at an early stage of downstream processing for the selective isolation of proteins. The technique utilizes a heterobifunctional ligand, which, in addition to having affinity for the target protein(s), possesses another function for controlling precipitation. The latter component is comprised of a polymer which can be made reversibly soluble and insoluble by altering a specific parameter such as pH or temperature. Different polymers of natural and synthetic origin have been used for this purpose. The soluble form of the ligand is used for the affinity binding step and precipitation is induced for obtaining separation of the affinity complex. Some of the polymers used in this laboratory include chitosan, alginate, Eudragit S-100 (copolymer of methacrylic acid and methyl methacrylate) and polyethyleneimine. Chitosan and alginate served as natural ligands for wheat germ agglutinin and pectinase, respectively. The aromatic dye Cibacron Blue 3GA coupled to Eudragit S 100 and polyethyleneimine way used for the affinity precipitation of some model enzymes such as lactate dehydrogenase and alcohol dehydrogenase. As prior removal of cell debris, etc., is essential for affinity precipitation, the possibility of integration of the technique with extraction in aqueous two-phase systems was also demonstrated.     

A FLASH of insight into cellular chemistry: genetically encoded labels for protein visualization in vivo

Genetically encoded fluorescent labels, such as green fluorescent protein, make it possible to visualize a protein's natural distribution and environment in living cells. A new approach to protein labeling in living cells has been devised in which a small, membrane-permeable ligand binds with high affinity and specificity to a short peptide motif that can be incorporated into the protein of interest; the ligand becomes brightly fluorescent after binding to the peptide.     

Reversible hydrogels from self-assembling artificial proteins

Recombinant DNA methods were used to create artificial proteins that undergo reversible gelation in response to changes in pH or temperature. The proteins consist of terminal leucine zipper domains flanking a central, flexible, water-soluble polyelectrolyte segment. Formation of coiled-coil aggregates of the terminal domains in near-neutral aqueous solutions triggers formation of a three-dimensional polymer network, with the polyelectrolyte segment retaining solvent and preventing precipitation of the chain. Dissociation of the coiled-coil aggregates through elevation of pH or temperature causes dissolution of the gel and a return to the viscous behavior that is characteristic of polymer solutions. The mild conditions under which gel formation can be controlled (near-neutral pH and near-ambient temperature) suggest that these materials have potential in bioengineering applications requiring encapsulation or controlled release of molecular and cellular species.     

Inhibition of assembly of bacterial cell division protein FtsZ by the hydrophobic dye 5,5'-bis-(8-anilino-1-naphthalenesulfonate)

To gain further insight into the structural relatedness of tubulin and FtsZ, the tubulin-like prokaryotic cell division protein, we tested the effect of tubulin assembly inhibitors on FtsZ assembly. Common tubulin inhibitors, such as colchicine, colcemid, benomyl, and vinblastine, had no effect on Ca2+-promoted GTP-dependent assembly of FtsZ into polymers. However, the hydrophobic probe 5, 5'-bis-(8-anilino-1-naphthalenesulfonate) (bis-ANS) inhibited FtsZ assembly. The potential mechanisms for inhibition are discussed. Titrations of FtsZ with bis-ANS indicated that FtsZ has one high affinity binding site and multiple low affinity binding sites. ANS (8-anilino-1-naphthalenesulfonate), a hydrophobic probe similar to bis-ANS, had no inhibitory effect on FtsZ assembly. Because tubulin assembly has also been shown to be inhibited by bis-ANS but not by ANS, it supports the idea that FtsZ and tubulin share similar conformational properties. Ca2+, which promotes GTP-dependent FtsZ assembly, stimulated binding of bis-ANS or ANS to FtsZ, suggesting that Ca2+ binding induces changes in the hydrophobic conformation of the protein. Interestingly, depletion of bound Ca2+ with EGTA further enhanced bis-ANS fluorescence. These findings suggest that both binding and dissociation of Ca2+ are capable of inducing FtsZ conformational changes, and these changes could promote the GTP-dependent assembly of FtsZ.     

Suramin is an active site-directed, reversible, and tight-binding inhibitor of protein-tyrosine phosphatases

The effect of suramin, a well known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers, on protein-tyrosine phosphatases (PTPases) has been examined. Suramin is a reversible and competitive PTPase inhibitor with Kis values in the low microM range, whereas the Kis for the dual specificity phosphatase VHR is at least 10-fold higher. Although suramin can also inhibit the activity of the potato acid phosphatase at a slightly higher concentration, it is 2-3 orders of magnitude less effective against the protein Ser/Thr phosphatase 1alpha and the bovine intestinal alkaline phosphatase. Suramin binds to the active site of PTPases with a binding stoichiometry of 1:1. Furthermore, when suramin is bound to the active site of PTPases, its fluorescence is enhanced approximately by 10-fold. This property has allowed the determination of the binding affinity of suramin for PTPases and several catalytically impaired mutant PTPases by fluorescence titration techniques. Thus, the active site Cys to Ser mutants bind suramin with similar affinity as the wild type, while the active site Arg to Ala mutant exhibits a 20-fold reduced affinity toward suramin. Interestingly, the general acid deficient Asp to Ala mutant PTPases display an enhanced affinity toward suramin, which is in accord with their use as improved "substrate-trapping" agents. That suramin is a high affinity PTPase inhibitor is consistent with the observation that suramin treatment of cancer cell lines leads to an increase in tyrosine phosphorylation of several cellular proteins. Given the pleiotropic effects of suramin on many enzyme systems and growth factor-receptor interactions, the exact in vivo actions of suramin require further detailed structure-activity investigation of suramin and its structural analogs.     

Effect of mutations on the sensitivity of human beta-cell glucokinase to liver regulatory protein

Human beta-cell glucokinase and its liver counterpart displayed a half-saturating concentration of glucose (S0.5) of about 8 mmol/l and a Hill coefficient of 1.7, and were as sensitive to inhibition by the rat liver regulatory protein as the rat liver enzyme. These results indicate that the N-terminal region of glucokinase, which differs among these three enzymes, is not implicated in the recognition of the regulatory protein. They also suggest that the regulatory protein, or a related protein, could modulate the affinity of glucokinase for glucose in beta cells. We have also tested the effect of several mutations, many of which are implicated in maturity onset diabetes of the young. The mutations affected the affinity for glucose and for the regulatory protein to different degrees, indicating that the binding site for these molecules is different. An Asp158 Ala mutation, found in the expression plasmid previously thought to encode the wild-type enzyme, increased the affinity for glucose by about 2.5-fold without changing the affinity for the regulatory protein. The mutations that were found to decrease the affinity for the regulatory protein (Asn166 Arg. Val203 Ala, Asn204 Gln, Lys414 Ala) clustered in the hinge region of glucokinase and nearby in the large and small domains. These results are in agreement with the concept that part of the binding site for the regulatory protein is situated in the hinge region of this enzyme.     

Characterization of metallothionein isoforms by reversed-phase high-performance liquid chromatography with on-line post-column acidification and electrospray mass spectrometric detection

Derivatization of the free cys3,4 in human albumin, which is reported to occur under physiological conditions, has been performed in vitro by reaction of the protein with ethacrynic acid. This modification has been investigated by mass spectrometry and circular dichroism. Ethacrynic acid has been proven to bind human albumin either covalently and non-covalently. This post-translational modification does not determine significant changes in the secondary structure of the protein, as shown by the comparable circular dichroism spectra of the native and the modified proteins. Furthermore, the binding properties of the human albumin samples have been investigated by circular dichroism and equilibrium dialysis. The affinity to the higher affinity binding sites does not change either for drugs binding to site I, like phenylbutazone, or to site II, like diazepam, while a small but significant increase has been observed for bilirubin, known to bind to site III. Nevertheless significant decreases of the affinity at the lower affinity binding sites of the modified protein were observed for both drugs binding to site I or to site II.     

Experimental and Monte Carlo simulation studies of the thermodynamics of polyethyleneglycol chains grafted to lipid bilayers

Experimental measurements of the affinity of binding of fluorescent acylated polyethyleneglycol (PEG) conjugates to bilayers containing varying levels of phosphatidylethanolamine-PEGs (PE-PEGs) have been combined with Monte Carlo simulations to investigate the properties of the polymer chains at a PEG-grafted lipid interface. The affinity of binding of such conjugates to large unilamellar phosphatidylcholine/phosphatidylethanolamine (9:1) vesicles decreases 27-fold as the size of the coupled PEG chain increases from 1 to 114 monomer units. Incorporation of increasing amounts of PE-PEG2000 or PE-PEG5000 into the vesicles progressively reduces the affinity of binding of acylpeptide-PEG2000 or -PEG5000 conjugates. Monte Carlo simulations of surfaces with grafted PEG chains revealed no significant dependence of several characteristic properties of the polymer chains, including the average internal energy per polymer and the radii of gyration, on the grafting density in the range examined experimentally. The average conformation of a surface-grafted PEG2000 or PEG5000 chain was calculated to be fairly extended even at low grafting densities, and the projected cross-sectional areas of the grafted PEG chains are considerably smaller than those predicted on the basis of the estimated Flory radius. The experimental variation of the binding affinity of acylated conjugates for bilayers containing varying mole fractions of PE-PEG2000 or -PEG5000 is well explained by expressions treating the surface-grafted PEG polymers either as a van der Waals gas or as a system of rigid discs described by scaled particle theory. From the combined results of our experimental and simulation studies we conclude that the grafted PEG chains exist in a "mushroom" regime throughout the range of polymer densities examined experimentally and that the diminished affinity of binding of acylated-PEG conjugates to bilayers containing PE-PEGs results from occlusion of the surface area accessible for conjugate binding by the mobile PE-PEG polymer chains.     

Cooperative MetR binding in the Escherichia coli glyA control region

We determined the relative binding affinity of the MetR protein for wild-type and mutant MetR binding sites 1 and 2 in the Escherichia coli glyA control region. The results show that MetR binding site 1 has a higher affinity for the MetR protein than binding site 2. In addition, the results suggest that binding of MetR to the glyA promoter is cooperative. Mutations that decrease the ability of MetR to bind to either site 1 or site 2 have no significant effect on MetR's ability to bend DNA.     

Active site modification of factor VIIa affects interactions of the protease domain with tissue factor

In the initiation of coagulation, tissue factor (TF) allosterically activates the serine protease factor VIIa (VIIa) through specific interactions with protease domain residues. These interactions, and consequently affinity for TF, may be influenced by conformational changes in the protease domain that result from zymogen-enzyme transition or occupancy of the active site by tight binding inhibitors. In functional competition and direct binding analysis, we determined affinities for zymogen and enzyme species of wild-type VII and of mutants at protease domain residues that contact TF. We demonstrate that TF binding is not influenced by zymogen activation, indicating that the protease domain of zymogen and enzyme dock similarly with TF. In contrast, active site occupancy enhanced the affinity for TF by predominantly decreasing the dissociation rate of the TF.VIIa complex. Of the three interface residues studied, only Met306 played a major role in the inhibitor-induced increase in affinity. Met306 is also important for transmitting the allosteric changes from TF to the active site, resulting in enhanced catalysis. This study thus provides evidence for a bidirectional conformational interdependence of the interface residue Met306 and the active site of VIIa.     

Ca2+ differentially regulates conventional protein kinase Cs' membrane interaction and activation

The regulation of conventional protein kinase Cs by Ca2+ was examined by determining how this cation affects the enzyme's 1) membrane binding and catalytic function and 2) conformation. In the first part, we show that significantly lower concentrations of Ca2+ are required to effect half-maximal membrane binding than to half-maximally activate the enzyme. The disparity between binding and activation kinetics is most striking for protein kinase C betaII, where the concentration of Ca2+ promoting half-maximal membrane binding is approximately 40-fold higher than the apparent Km for Ca2+ for activation. In addition, the Ca2+ requirement for activation of protein kinase C betaII is an order of magnitude greater than that for the alternatively spliced protein kinase C betaI; these isozymes differ only in 50 amino acids at the carboxyl terminus, revealing that residues in the carboxyl terminus influence the enzyme's Ca2+ regulation. In the second part, we use proteases as conformational probes to show that Ca2+dependent membrane binding and Ca2+-dependent activation involve two distinct sets of structural changes in protein kinase C betaII. Three separate domains spanning the entire protein participate in these conformational changes, suggesting significant interdomain interactions. A highly localized hinge motion between the regulatory and catalytic halves of the protein accompanies membrane binding; release of the carboxyl terminus accompanies the low affinity membrane binding mediated by concentrations of Ca2+ too low to promote catalysis; and exposure of the amino-terminal pseudosubstrate and masking of the carboxyl terminus accompany catalysis. In summary, these data reveal that structural determinants unique to each isozyme of protein kinase C dictate the enzyme's Ca2+-dependent affinity for acidic membranes and show that, surprisingly, some of these determinants are in the carboxyl terminus of the enzyme, distal from the Ca2+-binding site in the amino-terminal regulatory domain.     

Tight binding of bulky fluorescent derivatives of adenosine to the low affinity E2ATP site leads to inhibition of Na+/K+-ATPase. Analysis of structural requirements of fluorescent ATP derivatives with a Koshland-Némethy-Filmer model of two interacting ATP sites

A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na+/K+-ATPase (EC 3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321). The present work demonstrates that this model adequately describes all types of interactions and kinetics of a number of ATP analogs that differ in their cooperativity of the high and low affinity ATP binding sites of the enzyme. 2',3'-O(2,4,6-trinitrophenyl)ATP binds in a negative cooperative way to the E1ATP site (Kd = 0.7 microM) and to the E2ATP site (Kd = 210 microM), but 3'(2')-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E1ATP binding site (Kd = 200 microM) than to the E2ATP binding site (Kd = 80 microM). 3'(2')-O(5-Fluor-2,4-dinitrophenyl)-ATP, however, binds in a noncooperative way, with equal affinities to both ATP binding sites (Kd = 10 microM). In a research for the structural parameters determining ATP site specificity and cooperativity, we became aware that structural flexibility of ribose is necessary for catalysis. Moreover, puckering of the ring atoms in the ribose is essential for the interaction between ATP sites in Na+/K+-ATPase. A number of derivatives of 2'(3')-O-adenosine with bulky fluorescent substitutes bind with high affinity to the E2ATP site and inhibit Na+/K+-ATPase activity. Evidently, an increased number of interactions of such a bulky adenosine with the enzyme protein tightens binding to the E2ATP site.     

Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C

Mature protein kinase C is phosphorylated at a conserved carboxyl-terminal motif that contains a Ser (or Thr) bracketed by two hydrophobic residues; in protein kinase C betaII, this residue is Ser-660 (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This contribution examines how negative charge at this position regulates the function of protein kinase C. Specifically, Ser-660 in protein kinase C betaII was mutated to Ala or Glu and the enzyme's stability, membrane interaction, Ca2+ regulation, and kinetic parameters were compared with those of wild-type protein phosphorylated at residue 660. Negative charge at this position had no significant effect on the enzyme's diacylglycerol-stimulated membrane interaction nor the conformational change accompanying membrane binding. In contrast, phosphate caused a 10-fold increase in the enzyme's affinity for Ca2+ and a comparable increase in its affinity for phosphatidylserine, two interactions that are mediated by the C2 domain. Negative charge also increased the protein's thermal stability and decreased its Km for ATP and peptide substrate. These data indicate that phosphorylation at the extreme carboxyl terminus of protein kinase C structures the active site so that it binds ATP and substrate with higher affinity and structures determinants in the regulatory region enabling higher affinity binding of Ca2+. The motif surrounding Ser-660 in protein kinase C betaII is found in a number of other kinases, suggesting interactions promoted by phosphorylation of the carboxyl terminus may provide a general mechanism for stabilizing kinase structure.     

The binding of phenol red to rabbit renal cortex

The binding of phenol red to the microsomal fraction of rabbit kidney cortex was rapid, reversible and consisted of two independent populations of binding sites: a high affinity and low capacity class which had an association constant of 11.29 - 10(3) M-1 and a binding capacity of 2.41 mumol phenol red bound per g of protein, and a low affinity binding population with an association constant of 0.80 - 10(3) M-1 and a maximal binding capacity of 55.06 mumol per g of protein. Probenecid (0.32 mM) competitively inhibited phenol red binding to only the high affinity binding site, whereas 2,4-dinitrophenol (0.77 mM) competitively inhibited phenol red binding to both the high and the low affinity population of binding sites. The binding of phenol red was highly sensitive to the cationic composition of the medium. The affinity of phenol red to the high and the low affinity binding populations was lowered by decreasing the sodium and potassium concentrations to 19 and 6 mequiv./l, respectively; however, the maximal binding capacity was unchanged. Calcium appeared to have no effect on the phenol red binding to the microsomes. All of these considerations suggest that the high affinity phenol red binding to the microsomal fraction may represent the interaction of phenol red with the physiological receptor necessary for organic acid transport at the peritubular membrane. Phenol red binding to the low affinity binding population may indicate an intracellular binding population which contributes to the intracellular accumulation of weak organic acids.     

Preparations of psi-peptide bond and peptide-aldehyde inhibitors of atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor

Pseudo-peptide bond inhibitors (psi-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The psi-bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR-psi-LR and Bz-APR-psi-SLRR can be considered "readthrough inhibitors" of atrial granule serine proteinase. The most potent psi-peptide, Bz-APR-psi-SLRR (IC50=250 microM), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the psi-bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The psi-bond peptides containing two C-terminal Arg residues are three- to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the psi-peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands.     

Anomalous temperature dependence of the activity of immobilized alpha-chymotrypsin preparations

Catalytic activity of alpha-chymotrypsin preparations covalently included in the matrix of the poly-N-isopropylacrylamide gel does not follow Arrhenius equation above the low critical temperature of the polymer dissolution. Starting from this temperature, at which the changes of polymer structure takes place (hydrophobization), the temperature increase results in a rate lowering for the chemical reaction catalyzed by the enzyme. This phenomenon is reversible. A correlation between temperature dependence of the immobilized alpha-chymotrypsin activity and the dehydration degree of the carrier is observed. The decrease of the water content in the matrix causes a change of the substrate specificity of the immobilized alpha-chymotrypsin.