Anion-induced Conformational Changes Research Articles

PH- and chaotropic anion-induced conformational changes of tertiary amine-containing binary heterografted star molecular bottlebrushes in aqueous solution

Three-arm star-shaped, tertiary-amine-containing bottlebrushes exhibit star-globule shape transitions in response to pH changes and addition of sufficiently strong chaotropic anions.

Rotaxane and Catenane Host Structures for Sensing Charged Guest Species

CONSPECTUS: The promise of mechanically interlocked architectures, such as rotaxanes and catenanes, as prototypical molecular switches and shuttles for nanotechnological applications, has stimulated an ever increasing interest in their synthesis and function. The elaborate host cavities of interlocked structures, however, can also offer a novel approach toward molecular recognition: this Account describes the use of rotaxane and catenane host systems for binding charged guest species, and for providing sensing capability through an integrated optical or electrochemical reporter group. Particular attention is drawn to the exploitation of the unusual dynamic properties of interlocked molecules, such as guest-induced shuttling or conformational switching, as a sophisticated means of achieving a selective and functional sensor response. We initially survey interlocked host systems capable of sensing cationic guests, before focusing on our accomplishments in synthesizing rotaxanes and catenanes designed for the more challenging task of selective anion sensing. In our group, we have developed the use of discrete anionic templation to prepare mechanically interlocked structures for anion recognition applications. Removal of the anion template reveals an interlocked host system, possessing a unique three-dimensional geometrically restrained binding cavity formed between the interlocked components, which exhibits impressive selectivity toward complementary anionic guest species. By incorporating reporter groups within such systems, we have developed both electrochemical and optical anion sensors which can achieve highly selective sensing of anionic guests. Transition metals, lanthanides, and organic fluorophores integrated within the mechanically bonded structural framework of the receptor are perturbed by the binding of the guest, with a concomitant change in the emission profile. We have also exploited the unique dynamics of interlocked hosts by demonstrating that an anion-induced conformational change can be used as a means of signal transduction. Electrochemical sensing has been realized by integration of the redox-active ferrocene functionality within a range of rotaxane and catenanes; binding of an anion perturbs the metallocene, leading to a cathodic shift in the ferrocene/ferrocenium redox couple. In order to obtain practical sensors for target charged guest species, confinement of receptors at a surface is necessary in order to develop robust, reuseable devices. Surface confinement also offers advantages over solution based receptors, including amplification of signal, enhanced guest binding thermodynamics and the negation of solubility problems. We have fabricated anion-templated rotaxanes and catenanes on gold electrode surfaces and demonstrated that the resulting mechanically bonded self-assembled monolayers are electrochemically responsive to the binding of anions, a crucial first step toward the advancement of sophisticated, highly selective, anion sensory devices. Rotaxane and catenane host molecules may be engineered to offer a superior level of molecular recognition, and the incorporation of optical or electrochemical reporter groups within these interlocked frameworks can allow for guest sensing. Advances in synthetic templation strategies has facilitated the synthesis of interlocked architectures and widened their interest as prototype molecular machines. However, their unique host-guest properties are only now beginning to be exploited as a sophisticated approach to chemical sensing. The development of functional host-guest sensory systems such as these is of great interest to the interdisciplinary field of supramolecular chemistry.

Fluoride-Triggered ESPT in the Binding with Sal(oph)en

In this paper, anion binding and sensing affinity of the simple and easy-to-make salen, a typical class of ligand used comprehensively in metal coordination, was investigated. Results indicated that salophen was both a colorimetric and fluorescent selective chemosensor for fluoride ion, which operated by the anion-induced conformational changes and subsequently excited-state intramolecular proton transfer (ESPT) process. The F(-)-induced quick response, as well as noticeable optical changes, suggested that anion-sensing mechanism maybe help to design and to synthesize the new preferential selective probes for F(-).

Anion-induced conformational changes in 2,7-disubstituted indole-based receptors

The conformational preorganization and anion-induced conformational changes of indole-based receptors functionalized with an amide group at the 2-position and a variety of amide, urea and thiourea moieties at the 7-position have been studied by the means of NMR spectroscopy. NOE experiments showed that anti-anti orientation across C2-C2alpha and C7-N7alpha bonds is preferred for receptors 1-4 in acetone solution in the absence of anions. Anion-receptor interactions have been evaluated through (1)H and (15)N chemical shift changes. In 2,7-bis-carboxamido functionalized indoles the interaction with chloride and bromide anions primarily occurs at the indole H1 proton. The introduction of urea and thiourea moieties increases the number of hydrogen bond donor sites which manifests itself in a distribution of halide-receptor interactions among the H1, H7alpha and H7gamma protons. Acetate anions also interact strongly with indole and urea NH donor groups, whereas nitrate anions interact solely with H7alpha and H7gamma urea/thiourea protons. NOE enhancements in the presence of anions revealed that anion-receptor complexes favour the syn-syn conformation of the C2 and C7 substituents.

Interactions between Impermeant Blocking Ions in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore: Evidence for Anion-Induced Conformational Changes

It is well known that extracellular Cl(-) ions can weaken the inhibitory effects of intracellular open channel blockers in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. This effect is frequently attributed to repulsive ion-ion interactions inside the pore. However, since Cl(-) ions are permeant in CFTR, it is also possible that extracellular Cl(-) ions are directly competing with intracellular blocking ions for a common binding site; thus, this does not provide direct evidence for multiple, independent anion binding sites in the pore. To test for the possible through-space nature of ion-ion interactions inside the CFTR pore, we investigated the interaction between impermeant anions applied to either end of the pore. We found that inclusion of low concentrations of impermeant Pt(NO(2))(4) (2-) ions in the extracellular solution weaken the blocking effects of three different intracellular blockers [Pt(NO(2))(4) (2-), glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoic acid] without affecting their apparent voltage dependence. However, the effects of extracellular Pt(NO(2))(4) (2-) ions are too strong to be accounted for by simple competitive models of ion binding inside the pore. In addition, extracellular Fe(CN)(6) (3-) ions, which do not appear to enter the pore, also weaken the blocking effects of intracellular Pt(NO(2))(4) (2-) ions. In contrast to previous models that invoked interactions between anions bound concurrently inside the pore, we propose that Pt(NO(2))(4) (2-) and Fe(CN)(6) (3-) binding to an extracellularly accessible site outside of the channel permeation pathway alters the structure of an intracellular anion binding site, leading to weakened binding of intracellular blocking ions.

Anion- and pH-dependent activation of the soluble form of dopamine beta-hydroxylase.

The present study presents evidence for the conclusion that the catalytic activity of dopamine beta-hydroxylase (DBH; dopamine beta-mono-oxygenase, EC 1.14.17.1) is regulated independently by pH and by anions. In the absence of activating anions (i.e. in 50 mM Mes buffer) the activity was essentially zero at low pH (5.1-5.3) when assayed with the artificial electron donors ferrocyanide (0.25 mM), N, N, N', N'-tetramethyl-p-phenylenediamine (TMPD, 2 mM) or N, N-dimethyl-p-phenylenediamine (1 mM) and tyramine (8 mM) as the substrate to be hydroxylated. However, in the presence of activating anions (e.g. 0.05-0.6 M Cl(-) in 50 mM Mes buffer, 0.1 M phosphate buffer or 0.2 M acetate buffer) a high catalytic activity was observed at pH 5.1-5.3. The pronounced effect of anions at this pH may be related to the postulated anion-induced conformational change of DBH [Syvertsen, Melø and Ljones (1987) Biochim. Biophys. Acta 914, 6-18] resulting in a facilitated access of the substrates to the active site(s). The anion-activated DBH was inhibited when assayed with ferrocyanide and activated when assayed with TMPD as electron donors by increasing the pH (5.1 to 6.0). By contrast, in the absence of anions the activity increased from essentially zero at pH 5.1-5.3 to high values at pH 6.0, irrespective of the electron donor used. The data suggest that the conformational change induced by anion activation exposes a negatively charged group at or near the electron-donor-binding site(s) imposing an electrostatic repulsion towards ferrocyanide (four negative charges) and an electrostatic attraction towards the positively charged TMPD, thus explaining the different pH-activity curves obtained for the two electron donors. In contrast to the artificial electron donors, the physiological donor ascorbate [Terland and Flatmark (1975) FEBS Lett. 59, 52-56] supports hydroxylation of tyramine at low pH also in the absence of Cl(-), acetate or phosphate, confirming that ascorbate also functions as an anion activator.

Control of antibody-antigen interaction using anion-induced conformational change in antigen peptide.

The binding of a monoclonal antibody to an epitope peptide was controlled by the conformational change of the epitope peptide induced by anions. We synthesized peptides in which the epitope sequence DTYRYI for the monoclonal antibody AU1 is located between amphiphilic peptides (KKLL)n and (LLKK)n. In the absence of an appropriate anion, the peptide was in a random coil state and the epitope was linear. In contrast, in the presence of an appropriate anion, the peptide exhibited an anti-parallel alpha-helical structure and the epitope was subsequently 'bent'. In the presence of 41 microM triphosphate, the association constant between the antibody and the peptide was decreased by one order of magnitude in the case of n = 3 and at least three orders of magnitude in the case of n = 4 or 5. Oligo-DNAs, as anions, dissociated the antibody-peptide complex, whereas triphosphate did not. The DNA concentrations required for 50% dissociation of the antibody-peptide complex at pH 7.5 were 4x10(-8), 1x10(-7) and 6x10(-6) M for decamer, octamer and hexamer DNA, respectively.

Catalytic Properties of the Two Active Sites of Angiotensin I-Converting Enzyme on the Cell Surface

Angiotensin I converting enzyme is a zinc metallopeptidase that contains two very similar domains, each with an active site. Enzymatic studies of these active sites have always been performed on solubilized enzyme, although angiotensin I converting enzyme is a transmembrane ectopeptidase. The availability of transfected CHO cells expressing wild-type recombinant enzyme and mutants in which one of the two active sites has been inactivated by site-directed mutagenesis allowed the properties of each active site on the cell surface and the effect of anchorage and membrane environnement to be studied. Both active centers are catalytically active in the cell membrane-anchored enzyme and convert angiotensin I to angiotensin II. Comparison of the kinetic parameters for the transfected cells with those for the purified enzymes reveals differences in Kcat but suggests that no major conformational changes of these active sites occur upon anchorage of the enzyme to the cell membrane. The chloride activation profiles show that the two domains in the cell-bound enzyme also undergo the same anion-induced conformational changes as in the solubilized enzyme.

Anion-induced conformational change of apo-electron-transferring flavoprotein.

Apoprotein of electron-transferring flavoprotein (ETF) exists in an equilibrium between two different forms, only one of which can associate with FAD (Sato, K. et al. (1991) J. Biochem. 109, 734-740), as represented in the following kinetic scheme: A* in equilibrium with A, A+FAD in equilibrium with holoETF, where "A*" and "A" are the different forms of apoETF. In the present study, the effects of various anions on the conversion between the two forms of apoETF were investigated by kinetic analyses on binding of FAD to apoETF. All the anions tested here induced the conversion from "A*" to "A"; the order of the effectiveness was I- approximately Br- greater than Cl- greater than F-. Further, glycerol also induced the conversion from "A*" to "A". The elution pattern of apoETF on molecular sieve chromatography was changed by addition of salts or glycerol; this change was due to the conversion from "A*" to "A" by the added solutes. The "A*" form was eluted more rapidly than the "A" form, indicating that the "A*" form exists in a looser conformation than the "A" form. The far-UV CD spectral change upon addition of salts indicated that a greater part of the secondary structure is retained in the conversion from "A*" to "A," but the "A" form contains a somewhat larger amount of beta-sheet than "A*."

The effect of zinc on the activity and fluorescence of carbonic anhydrase holoenzymes.

The addition of Zn2+ to human carbonic anhydrase B holoenzyme was shown to enhance the protein fluorescence, and this enhancement was correlated with the inhibition of the p-nitrophenyl acetate esterase activity. The affinity for the inhibitory Zn2+ was increased when the ionic inhibitors, acetate or chloride, were added, suggesting that the inhibitory Zn2+-binding site is within the region of the protein that undergoes an anion-induced conformational change. A similar fluorescence enhancement was observed when Zn2+ was added to human carbonic anhydrase C and to bovine carbonic anhydrase, demonstrating that the binding site is not a thiol group. Circular-dichroism studies showed that the C isoenzyme but not the B isoenzyme underwent a major conformational change in the presence of Zn2+. A mechanism for the Zn2+-induced fluorescence enhancement was suggested on the basis of studies with simple compounds.

Dopamine-β-hydroxylase: THE SUBUNIT STRUCTURE AND ANION ACTIVATION OF THE BOVINE ADRENAL ENZYME

Abstract Bovine adrenal medulla 3,4-dihydroxyphenylethylamine β-hydroxylase has been further characterized. The enzyme consists of four subunits, two pairs of which are linked by disulfide bridges and two pairs of which are held together by noncovalent bonds. Preliminary evidence suggests that these subunits are identical. The activation of this enzyme by anions has been studied. The binding of anions to a basic group adjacent to the ionizable group at the active site has been proposed to explain the mechanism of activation by these compounds. No evidence has been found for an anioninduced conformational change.