Intramolecular Salt Research Articles

On the role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II.

The role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II has been investigated by means of site-directed mutagenesis and cross-linking. Replacement of Asp-9 resulted in a dramatic increase in proteolytic sensitivity leading to the degradation of the protein forming a 31 kDa fragment with an undefined N-terminus. This fragment was unable to restore oxygen evolution. However, the variants of the 33 kDa protein which remained intact could reconstitute oxygen evolution as effectively as the wild-type protein. Cross-linking experiments with a water-soluble carbodiimide revealed that mutagenesis of residue D9 led to the disruption of an intramolecular salt bridge. Therefore we suggest that the N-terminus of the 33 kDa protein is necessary for maintaining the binding ability of the protein to Photosystem II but might not be involved in binding itself.

Ionic interactions in crystalline bovine pancreatic ribonuclease A.

Isomorphous crystals (space group P3(2)21) of bovine pancreatic ribonuclease A (RNase A) were prepared at a pH of 5.5 in a series of high salt conditions, where both the nature of the ions and the ionic strength varied: 80% ammonium sulfate (mu = 12.5); 8 M sodium formate (mu = 8.0); 3 M NaCl, 30% ammonium sulfate (mu = 7.0); 3 M CsCl, 30% ammonium sulfate (mu = 7.0); and 2.5 M NaCl, 3.3 M sodium formate (mu = 5.8). These structures were independently refined to a resolution of 2.0 A or better with R-factors that range from 16.1% to 17.5%. A comparison of these six structures and the monoclinic crystal form of RNase A grown from alcohol shows that changes in ionic strength do not alter the secondary or tertiary structure and that there are no significant changes in intramolecular salt bridges. These findings support the notion that structures determined from crystals grown in high salt are representative of the overall structural and electrostatic features present under physiological conditions. While little effect was observed on the main chain conformation, several residues adopted different side chain conformations and altered hydrogen-bonding patterns, either as result of direct anion binding or more subtle indirect effects. Changes in the ionic composition of the mother liquor allowed for the occupancy of the active site with different anions. The direct observation of active site-bound chloride and formate anions supports the proposal that these species act as true competitive inhibitors of RNase A and not through nonspecific electrostatic effects. The identification of bound formate anions allowed for an experimental validation of computational-based functional group mapping techniques and suggests a useful modification to these approaches. Electrostatic surface potential calculations identify a nearly continuous band of positive potential, consistent with an extended binding site for polynucleotide ligands and substrates. The majority of these residues are not involved in salt bridges, which may facilitate binding to extended polynucleotide substrates. Selection of the appropriate solvent conditions results in an unoccupied active site, which will allow this crystal form to be used for the crystallographic study of productive ligand-binding modes.

Importance of highly conserved anionic residues and electrostatic interactions in the activity and structure of the cardiotonic polypeptide anthopleurin B.

Several polypeptide toxins from sea anemones caused delayed inactivation of mammalian voltage-dependent sodium channels, resulting in a positive inotropic effect on the heart. Anthopleurin B (ApB), a toxin produced by the sea anemone Anthopleura xanthogrammica, is the most potent of all known anemone toxins. Previous studies in this laboratory have both defined and revealed an important role for the cationic cluster of Arg-12, Arg-14, and Lys-49 in the expression of ApB's biological activity. In the present investigation, we explore the role of all remaining charged residues by producing and characterizing mutants of ApB at Asp-7, Asp-9, Lys-37, His-39, and His-34. Recombinant toxins have been purified to homogeneity and their abilities to enhance veratridine-dependent sodium uptake in cell lines expressing either the neuronal or cardiac isoform of the sodium channel evaluated. Replacement of Asp-7 results in a product that fails to fold, while muteins H39A and H34A have activities very similar or identical to wild-type ApB. In contrast, the D9N and K37A muteins are 7-12-fold less active that wild-type ApB, and truncation of the side chain in D9A results in a further decrease in activity, especially in the cardiac model. We conclude that although a negative charge per se is not essential at position 9, the presence of a hydrogen-bond forming side chain is critical both for appropriate folding and for interaction with the sodium channel. Because the K37A and H39A mutant toxins can fold normally, neither Lys-37 nor His-39 seem to participate in an intramolecular salt bridge, in contrast to suggestions arising from NMR studies of ApA and ApB. However, Lys-37 may play a role in channel interaction.

Intramolecular donor-stabilized phosphanylium salts via λ3-Functionalized-iminophosphanes

λ 3-Iminophosphanes, R-P=NAr ∗ ( Ar ∗=2,4,6-tri-tBuC 6H 2 ),containing intramolecularly coordinating substituents at the dicoordinate phosphorus atom (R=8-NMe 2 C 10H 6 or 2-Ph 2PCH 2 C 6H 4 have been synthesized and used for generation of the isolable phosphanylium salts stabilized by a donor P→ P bond.

A single point chiral inversion that selforganizes a randomcoil peptide. Apolar solvent conformation of Boc-( L\\ D)-Glu-Ala-Leu-LysNHMe

The tetrapeptide Boc- L-Glu-Ala-Leu-LysNHMe ( 1) reveals a random coil conformation, based on its Glu(γ) and Lys(ɛ) methylene proton aniosotropic shift, GluNH chemical shift, NOEs in chloroform-DMSO (6:1), and its amide proton temperature coefficients in DMSO, while on similar considerations, the diastereomer Boc- D-Glu-Ala-Leu-LysNHMe ( 2) is characterized as a highly ordered 3/10 type distorted protohelix with a remarkably stable intramolecular salt bridge under these solvent conditions.

Circular Dichroism Study on Fully Bioactive CCK-Peptides of Increasing Chain Length

A CD conformational analysis has been performed on CCK-peptides elongated at the N-terminus in sequence mode beyond the naturally occurring CCK-8 up to the pentadecapeptide sequence. By extending N-terminally the CCK-8 sequence an intramolecular salt bridge between the tyrosine-O-sulfate and the arginine guanido function is allowed to be established. However, this intramolecular electrostatic interaction was not found to affect the bioactivities of the CCK-peptides indicating that induction of such salt bridge at the level of the ligand molecule does not prevent a similar interaction at receptor level by exchange of the counterion partner. As expected for unconstrained short linear peptides the dichroic properties in aqueous solution were indicative of predominantly random coil structure. Conversely, in aqueous TFE the CD spectra were consistent with the presence of γ-type turns similarly to what has been observed under identical conditions for small size peptides related to the homologuous gastrin hormone. In surfactant solutions the CCK-peptides were found to assume β-type structures by inserting at least the C-terminal portion of the bioactive core into more hydrophobic compartments of the surfactant micelles, whereas the hydrophilic charged N-termini of the CCK-pep-tides of increasing chain length are exposed to the water phase in random coil structures as suggested by the CD spectra. This contrasts previous findings related to the homologuous gastrin peptides, where identical CD spectra were recorded in aqueous TFE and in presence of micelles. This observation strongly suggests that gastrin and CCK related peptides exhibit distinct conformational preferences, despite their high degree of sequence homology, and fully agrees with the ability of CCK to interact specifically with different receptors

Synthetic pH sensitive polyampholyte hydrogels: A preliminary study

A combined capillary drop viscometer-pH cell has been constructed and used to study the solution properties of some polymeric weak acids, bases, and ampholytes containing methacrylic acid (MAA), dimethylaminoethyl methacrylate (DMAEMA), and 2-hydroxyethyl methacrylate (HEMA). The behavior of polyelectrolytes based on MAA or DMAEMA is consistent with established thinking, and is dominated by the chain expansions which accompany ionization of acidic (MAA) or protonation of basic (DMAEMA) functionality. The solution behavior of the polyampholytes is complex and was studied as a preliminary to investigations of crosslinked hydrogel analogues. Briefly, it was found that changes in polymer coil dimensions were dictated by the formation or “breakage” of intramolecular salt bridges by, for example, lowering the solution pH to protonate weakly acidic, or raising the pH to deprotonate weakly basic functionality. Similar effects were observed on adding counterions which preferentially complex with one of the bound ions. The pH “sensitivity” of polymer coil dimensions increased with the concentration of charged functionality in the polymer. Chemically crosslinked polyampholytes were prepared and equilibrium water contents were measured as a function of pH. In contrast, due to network constraints, the pH “sensitivity” of the hydrogels, as measured by changes in equilibrium water uptake, increased with a decrease in the level of charged functionality in the polymer.

Actions of chiriquitoxin on frog skeletal muscle fibers and implications for the tetrodotoxin/saxitoxin receptor.

Chiriquitoxin (CqTX) from the Costa Rican frog Atelopus chiriquensis differs from tetrodoxin (TTX) only in that a glycine residue replaces a methylene hydrogen of the C-11 hydroxymethyl function. On the voltage-clamped frog skeletal muscle fiber, in addition to blocking the sodium channel and unrelated to such an action, CqTX also slows the activation of the fast potassium current in approximately 40% of the muscle fiber population. At pH 7.25, CqTX is as potent as TTX in blocking the sodium channel, with an ED50 of 3.8 nM. Its ED50's at pH 6.50 and 8.25 are 6.8 and 2.3 nM, contrasted with 3.8 and 4.3 nM for TTX. These differences are attributable to changes in the chemical states in the glycine residue. The equipotency of CqTX with TTX at pH 7.25 is explainable by an intramolecular salt bridge between the amino and carboxyl groups of the glycine function, all other surface groups in TTX and CqTX being the same. From available information on these groups and those in saxitoxin (STX), the TTX/STX binding site is deduced to be in a pocket 9.5 A wide, 6 A high, and 5 A deep. The glycine residue of CqTX probably projects out of the entrance to this pocket. Such a view of the binding site could also account for the actions of STX analogues, including the C-11 sulfated gonyautoxins and the 21-sulfocarbamoyl analogues. In the gonyautoxins the sulfate groups are equivalently placed as the glycine in CqTX, whereas in the sulfocarbamoyl toxins the sulfate groups extend the carbamoyl side-chain, leading to steric hinderance to productive binding.

Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding.

In order to identify residues required for the binding of interleukin-8 (IL-8) to its receptor, mutants were constructed in which clusters of charged amino acids were systematically replaced with alanine along the entire IL-8 sequence. The mutants were tested for their ability to induce a receptor-mediated rise in cytosolic free Ca2+, a property of wild-type IL-8 which can readily be detected by flow cytometry using neutrophils loaded with the calcium probe Indo-1. Eleven of the 12 mutants caused neutrophil calcium mobilization at 5 nM; the exception being a triple alanine mutant at positions K3, E4, and R6, which was inactive at all concentrations tested (150 nM maximum). A second set of mutants was generated in which residues 1-15 were individually mutated to alanine. Mutants E4A, L5A, or R6A were all inactive in the Ca2+ assay at 5 nM and competed poorly with 125I-IL-8 for neutrophil receptor binding; I10A, E4A, L5A, and R6A had approximately 30-, 100-, 100-, and 1000-fold reduced affinity, as compared with control IL-8, respectively. The nuclear magnetic resonance structure of IL-8 indicates that, in solution, the side chains of E4, L5, R6, and I10 point away from the core of the protein and do not participate in any intramolecular hydrogen bonds or salt bridges (Clore, G. M., and Gronenborn, A. M. (1991) J. Mol. Biol. 217, 611-620).

Chemistry of synthetic receptors and functional group arrays. 17. The intramolecular salt effect. An acrylate ester bearing an ion pair shows enhanced rates and stereoselectivity in a nitrone cycloaddition

Chemistry of synthetic receptors and functional group arrays. 17. The intramolecular salt effect. An acrylate ester bearing an ion pair shows enhanced rates and stereoselectivity in a nitrone cycloaddition

The complementary redox properties of viologens and pyromellitimides: a new class of organic conductors

Organic charge-transfer (CT) salts have been of interest for some years, especially since the discovery of the metallic conductivity of the TTF-TCNQ salt. The authors have begun an investigation of the charge-transfer chemistry of viologens and pyromellitimides, based on their complementary redox properties, with the intention of proceeding from intermolecular examples to intramolecular CT salts and finally to examples in which the viologens and pyromellitimides are incorporated in a polymer. They report progress in the first two stages of this investigation, including isolation of a new, highly conductive intermolecular CT salt and electrochemical characterization of a covalently linked viologen/pyromellitimide. These studies point to an interesting new class of organic solid-state materials.

The chemistry of functional group arrays. Electrostatic catalysis and the “intramolecular salt effect”.

Ionic groups and salt bridges in the active sites of enzymes are thought to have important effects upon substrate reactivity. This paper describes the evaluation of an ion pair as an intramolecular effector of reaction rate for an addition reaction. The results reveal that in chloroform the intramolecular salt effect can lead to very large rate enhancements. It is proposed that in this case the local effect of the ion pair probably depends upon two factors: the electrostatic effects of the salt pair and, to a lesser extent, an intramolecular hydrogen bond. This observation of large rate enhancements in nonpolar solvents provides evidence of the substantial effects that ion pairs within the active sites of enzymes could have on reaction rates. The most specific and strongest synthetic molecular associations have been reported for host-guest systems in chloroform, and it is foreseen that hydrogen bond based host-guest systems will be combined with the intramolecular salt effect to bring within reach a large class of new shape selective specific synthetic catalysts and molecular effectors.

The chemistry of synthetic receptors and functional group arrays. 13. The intramolecular salt effect

Ionic groups and salt bridges in the active sites of enzymes are thought to have important effects upon substrate reactivity. This paper describes the evaluation of an ion pair as an intramolecular effector of reaction rate for an addition reaction, the reaction of benzylic primary amines with methyl propynoate

Synthesis and acid ionization constants of cyclic cystine peptides H-Cys-(Gly)n-Cys-OH (n = 0-4).

Cyclic peptide disulfides of the general formula H-Cys-(Gly)n-Cys-OH (n = 0-4) were synthesized from the corresponding peptide derivatives [Boc-Cys(Trt)(Gly)-n-Cys(Trt)-OBut] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK1 = 2.35-2.84) and to terminal amino group (pK2 = 5.61-6.93); pK1 values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK2 values. These trends could be tentatively attributed to the intramolecular salt bridge (-COO- ----NH+3-) formation.

Strong participation of selenium substituents in decomposition of pyrazolines formed by 1,3-dipolar cycloaddition of appropriate vinyl selenides with diazoalkanes

Decomposition of 4-(arylseleno)pyrazolines (3) bearing two electron-withdrawing groups such as COOMe, COMe, and CN at C-3, which were prepared in situ by reaction of the corresponding aryl vinyl selenides (2) with diazoalkanes, are reported. The pyrazolines decompose below 0{degree}C to give allyl aryl selenide derivatives (4) by migration of 4-arylseleno group to C-5 concerted with extrusion of nitrogen. Facile decomposition of the pyrazolines with migration of the selenium substituent is explained by strong contribution of intramolecular diazonium salt resonance structure, within which arylseleno groups strongly participate in the decomposition. Most reactions of 2 with 2 mol of diazoalkanes gave the pyrazolines 5, whereas reaction of 2c with 2 mol of 2-diazopropane gave a reverse orientation adduct, the pyrazoline 9.

Structural variations in the alanine‐rich antifreeze proteins of the pleuronectinae

The sequence and activity of antifreeze proteins from two right eye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97-residue preproportion. Edman degradation identified the N-terminus of the 48-amino-acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5' and 3' untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11-amino-acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 11-amino-acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice-binding residues per molecule.

Peptide models of electrostatic interactions in proteins: NMR studies on two beta-turn tetrapeptides containing Asp-His and Asp-Lys salt bridges.

Two model peptides Boc-Asp-Pro-Aib-X-NHMe [X = His (1) and X = Lys (2)] were synthesized to simulate intramolecular electrostatic interactions between ionizable side chains. Conformational analysis by 270-MHz 1H NMR in (CD3)2SO reveals that the backbone secondary structures of these two peptides are stabilized by two strong intramolecular hydrogen bonds, involving the consecutive carboxy-terminal NH groups. 1H NMR chemical shifts were measured in 1, 2, and a protected derivative, Boc-Asp(OBzl)-Pro-Aib-His-NHMe (3). These shifts were also measured for the model compounds Ac-Lys-NHMe, Boc-Asp-NHMe, and Boc-His-NHMe in their different states of ionization. An analysis of the chemical shifts of the ionization-sensitive reporter resonances suggests the formation of a strong intramolecular salt bridge in the lysyl peptide 2 and a bridge of moderate strength in the histidyl peptide 1. A comparison of the temperature dependence of chemical shifts in peptides 1-3 suggests that intramolecular salt bridge formation results in diminished backbone flexibility. The results establish that proximity effects confer far greater stability to intramolecular ion pair interactions vis-a-vis their intermolecular counterparts. The salt bridge interaction in peptide 1 displays a remarkable sensitivity to the dielectric constant of the solvent medium. The results suggest that these peptides are good simulators of the role of salt bridges in the structural dynamics of proteins.

18] Stabilization and destabilization of protein structure by charge interactions

Protein molecules are unique polyelectrolytes and carry their own partial complement of counterions that exhibit charge cancellations at their characteristic isoionic point, and tight binding in the form of intramolecular salt bridges. When the protein's own charge array is sufficiently asymmetric by either composition, geometry, or both, the surface electrostatic potential in the absence of steric hindrance will dictate specific ion binding sites. Any calculation of protein stabilization as a function of pH and ionic strength include the phenomenon of specific ion binding where applicable. Electrostatic stabilization accounts for approximately half of the energy of dimer-tetramer assembly in hemoglobin; its role is marginal in the combination of trypsin with bovine pancreatic trypsin inhibitor (BPTI). Each protein studied shows a specific pattern of stabilization or destabilization resulting from a unique set of long-range, overlapping coulombic fields associated with each charge site. The individual contributions, and hence, the overall stabilization are affected as the array is modified by changes in pH, ionic strength, binding of charge components, or alteration of solvent exposure.

Reaction of pyridine N-imides with ylidenemalononitriles: formation of a novel intramolecular pyridinium salt

Pyridine N-imides react with ylidenemalononitriles to form a novel intramolecular pyridinium salt containing a dipole structure.

Non-choline-containing phospholipids solubilize divalent metal ions in organic solvents. The basis for a sensitive assay procedure.

Phospholipids which do not contain choline were found to solubilize Ni(II) in the chloroform phase resulting from the Folch extraction procedure. On the basis of this phenomenon a radioactive assay for such phospholipids was developed using 63Ni(II). This assay can be made rather sensitive by scaling down and can be performed relatively rapidly. The disadvantages are that it does not measure phosphatidyl chlorine or sphingomyelin and that different phospholipids bind different amounts of Ni(II). Alternatively, non-choline-containing phospholipids can be assayed colorimetrically on the basis of their ability to solubilize Cu(II) in the chloroform phase. The failure of phosphatidyl choline and sphingomyelin to bind divalent cations may be due to formation of an intramolecular salt between the negatively charged phosphate group and the positively charged nitrogen.