Ion-induced Conformational Changes Research Articles

19F-NMR Probing of Ion-Induced Conformational Changes in Detergent-Solubilized and Nanodisc-Reconstituted NCX_Mj.

Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.

Determining Ligand and Ion-Induced Conformational Changes in Serotonin Transporter with Its Fluorescent Substrates.

Conformational changes are fundamental events in the transport mechanism. The serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin after its release by serotonergic neurons and is the molecular target for antidepressant drugs and psychostimulants. Despite significant progress in characterizing the structure–function relationship of SERT, its conformational mechanism has not been fully understood. We present here a cell-based method for determining conformational changes in SERT with its fluorescent substrates by fluorescence imaging analysis. This method fluorometrically measures accessibility of strategically positioned cysteine residues in the substrate permeation pathway to calculate the rate constants of reactivity with MTS reagents in live or permeabilized cells. We validated this method by investigating ligand and ion-induced conformational changes in both the extracellular and cytoplasmic pathways of SERT. Furthermore, we applied this method for examining the influence of Cl− binding and vilazodone inhibition on SERT conformation. Our results showed that Cl− ion, in the presence of Na+, facilitates the conformational conversion from outward to inward open states, and that vilazodone binding stabilizes SERT in an outward open and inward-closed conformation. The present work provided insights into the conformational mechanism of SERT and also indicated that the cell-based fluorometric method is robust, straightforward to perform, and potentially applicable to any monoamine transporters in exploring the transport mechanism and mechanism of action of therapeutic agents for the treatment of several psychiatric disorders.

Incorporation and Utility of a Responsive Ribonucleoside Analogue in Probing the Conformation of a Viral RNA Motif by Fluorescence and 19 F NMR Spectroscopy.

Development of versatile probes that can enable the study of different conformations and recognition properties of therapeutic nucleic acid motifs by complementing biophysical techniques can greatly aid nucleic acid analysis and therapy. Here, we report the design, synthesis and incorporation of an environment-sensitive ribonucleoside analogue, which serves as a two-channel biophysical platform to investigate RNA structure and recognition by fluorescence and 19 F NMR spectroscopy techniques. The nucleoside analogue is based on a 5-fluorobenzofuran-uracil core and its fluorescence and 19 F NMR chemical shifts are highly sensitive to changes in solvent polarity and viscosity. Notably, the modified ribonucleotide and phosphoramidite substrates can be efficiently incorporated into RNA oligonucleotides (ONs) by in vitro transcription and standard solid-phase ON synthesis protocol, respectively. Fluorescence and 19 F readouts of the nucleoside incorporated into model RNA ONs are sensitive to the neighbouring base environment. The responsiveness of the probe was aptly utilized in detecting and quantifying the metal ion-induced conformational change in an internal ribosome entry site RNA motif of hepatitis C virus, which is an important therapeutic target. Taken together, our probe is a good addition to the nucleic acid analysis toolbox with the advantage that it can be used to study nucleic acid conformation and recognition simultaneously by two biophysical techniques.

Alkali metal ion-induced conformation changes of methionine- and leucine enkephalin investigated by gas-phase hydrogen/deuterium exchange combined with theoretical calculations

We have investigated the effects of alkali-metal ions (G+ = K+, Rb+, or Cs+) on the conformation of leucine enkephalin (LE) and methionine enkephalin (ME) using hydrogen/deuterium exchange mass spectrometry and theoretical calculations at the molecular level. The MS result revealed a 1:1 stoichiometric ratio of the G+ coordination with LE/ME. We used [LE/ME+H]+ as the native conformation for comparison purposes, we observed different HDX behaviors following the reactivity order [LE/ME+H]+>[LE/ME+K]+>[LE/ME+Rb]+>[LE/ME+Cs]+ and [LE+G]+>[ME+G]+. Eight hydrogen atoms were HD-exchanged for [LE/ME+H]+, whereas only 4, 3, and 2 hydrogen atoms underwent HDX for [LE/ME+K]+, [LE/ME+Rb]+, and [LE/ME+Cs]+, respectively. Additionally, we obtained different dissociation processes and different fragmentations for the various complexes, and their varied HDX behaviors suggest that the alkali-metal ions K+, Rb+, and Cs+ produce effects on the LE/ME conformations that differ from those of H+. Further, we investigated the favorable conformations of the complexes by theoretical calculations, and found that K+, Rb+, and Cs+ are coordinated with three carbonyl oxygens and one deprotonated carboxylic acid of LE/ME, whereas the terminal amino group is the most stable site for protonation. We calculated the proton affinity (PA) for each active hydrogen atom on the optimized complexes, which reasonably explained the HDX results that a high PA value corresponds to low HDX reactivity. Overall, the calculations showed that alkali-metal ions can induce ME into a more rigid conformation than LE. Even the native conformation of ME is more exposed than LE, and the conformation of LE/ME is stabilized and rigid owing to the increase in the size of the complexed alkali metals.

S100A4: a novel partner for heat shock protein 47 in antler stem cells and insight into the calcium ion-induced conformational changes

S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma

Xeno Nucleic Acid Nanosensors for Enhanced Stability

The omnipresence of salts in biofluids creates a pervasive challenge in designing sensors suitable for in vivo applications. Fluctuations in ion concentrations have been shown to affect the sensitivity and selectivity of optical sensors based on single-walled carbon nanotubes wrapped with single-stranded DNA (ssDNA-SWCNTs). We herein observe fluorescence wavelength shifting for ssDNA-SWCNT-based optical sensors in the presence of divalent cations at concentrations above 3.5 mM. In contrast, no shifting was observed for concentrations up to 350 mM for sensors bioengineered with increased rigidity using xeno nucleic acids (XNAs). Transient fluorescence measurements reveal distinct optical transitions for ssDNA- and XNA-based wrappings during ion-induced conformation changes, with XNA-based sensors showing increased permanence in conformational and signal stability. This demonstration introduces synthetic biology as a complementary means for enhancing nanotube optoelectronic behaviour, unlocking previously unexplored possibilities for developing nano-bioengineered sensors with augmented capabilities.

(Invited) Near-Infrared Fluorescence Modulation of Refolded DNA Aptamer-Functionalized Single-Walled Carbon Nanotubes for Optical Sensing

Selective modulation of near-infrared (NIR) fluorescence of single-walled carbon nanotubes (SWNTs) is important for their applications as NIR optical sensors and devices. Here we present the target molecule-mediated NIR fluorescence modulation of refolded DNA aptamer-functionalized SWNTs, using platelet-derived growth factor (PDGF) and a PDGF-binding aptamer as a model system. The aptamer–SWNT complexes use SWNT as nanoscale NIR optical emitters and DNA aptamers as molecular recognition elements. The binding of target molecules, PDGFs in this study, to PDGF-binding aptamers on the surface of SWNTs induces a conformation change of the aptamers, which modulates the NIR fluorescence of SWNT emitters. This study suggests that PDGF-binding aptamers noncovalently assembled on the SWNT surface can undergo a temperature and divalent ion-induced conformational change into a folded structure through multiple stages, which renders aptamer-functionalized SWNTs optically responsive to target molecules. In addition, our experimental and theoretical results show that the aptamers have a nanotube diameter-dependent affinity for SWNTs. We demonstrate that refolded aptamer-functionalized SWNTs reversibly modulate their NIR fluorescence in response to PDGF at the nanomolar range of 0.1 to 10 nM with apparent dissociation constants of ~0.71 nM (solution-phase complexes) and ~3.1 nM (complexes in hydrogels). This study could open new opportunities to design label-free, reversible NIR optical sensors that can detect various target molecules.

Structure-dynamic and functional relationships in a Li+-transporting sodium‑calcium exchanger mutant

The cell membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers control Ca2+ homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na+ and Ca2+, whereas NCLX can exchange Ca2+ with either Na+ or Li+. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (Kint = 0.8–1.4), whereas the extracellular vestibule is predominantly accessible to ions (Kint = 0.1–0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the “Ca2+-site” (SCa) of NCLX_Mj binds Na+, Li+, or Ca2+, whereas one or more additional Na+/Li+ sites of NCLX_Mj are incompatible with the Na+ sites (Sext and Sint) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na+/Li+ (but not for Ca2+) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca2+ homeostasis in distinct sub-cellular compartments.

Xeno Nucleic Acid Nanosensors for Enhanced Stability Against Ion-Induced Perturbations.

The omnipresence of salts in biofluids creates a pervasive challenge in designing sensors suitable for in vivo applications. Fluctuations in ion concentrations have been shown to affect the sensitivity and selectivity of optical sensors based on single-walled carbon nanotubes wrapped with single-stranded DNA (ssDNA-SWCNTs). We herein observe fluorescence wavelength shifting for ssDNA-SWCNT-based optical sensors in the presence of divalent cations at concentrations above 3.5 mM. In contrast, no shifting was observed for concentrations up to 350 mM for sensors bioengineered with increased rigidity using xeno nucleic acids (XNAs). Transient fluorescence measurements reveal distinct optical transitions for ssDNA- and XNA-based wrappings during ion-induced conformation changes, with XNA-based sensors showing increased permanence in conformational and signal stability. This demonstration introduces synthetic biology as a complementary means for enhancing nanotube optoelectronic behavior, unlocking previously unexplored possibilities for developing nanobioengineered sensors with augmented capabilities.

In vitro reconstitution and biochemical characterization of human phospholipid scramblase 3: phospholipid specificity and metal ion binding studies.

Human phospholipid scramblase 3 (hPLSCR3) is a single pass transmembrane protein that plays a vital role in fat metabolism, mitochondrial function, structure, maintenance and apoptosis. The mechanism of action of scramblases remains still unknown, and the role of scramblases in phospholipid translocation is heavily debated. hPLSCR3 is the only member of scramblase family localized to mitochondria and is involved in cardiolipin translocation at the mitochondrial membrane. Direct biochemical evidence of phospholipid translocation by hPLSCR3 is yet to be reported. Functional assay in synthetic proteoliposomes upon Ca2+ and Mg2+ revealed that, apart from cardiolipin, recombinant hPLSCR3 translocates aminophospholipids such as NBD-PE and NBD-PS but not neutral phospholipids. Point mutation in hPLSCR3 (F258V) resulted in decreased Ca2+ binding affinity. Functional assay with F258V-hPLSCR3 led to ~50% loss in scramblase activity in the presence of Ca2+ and Mg2+. Metal ion-induced conformational changes were monitored by intrinsic tryptophan fluorescence, circular dichroism, surface hydrophobicity changes and aggregation studies. Our results revealed that Ca2+ and Mg2+ bind to hPLSCR3 and trigger conformational changes mediated by aggregation. In summary, we suggest that the metal ion-induced conformational change and the aggregation of the protein are essential for the phospholipid translocation by hPLSCR3.

Zinc ion-induced conformational changes in new Delphi metallo-β-lactamase 1 probed by molecular dynamics simulations and umbrella sampling.

The hydrolysis of a β-lactam core ring caused by new Delphi metallo-β-lactamase 1 (NDM-1) with the help of two zinc cofactors induces significant resistance toward β-lactam antibiotics. Molecular dynamics (MD) simulations and the umbrella sampling method are integrated to study the conformational change mechanism of NDM-1 mediated by zinc ion binding. The statistical analyses of interaction contacts of the antibiotic ampicillin (AMP) with residues based on MD trajectories suggest that two Zn ions are essential for maintaining the binding of AMP with NDM-1. Umbrella sampling simulations further reveal that double-Zn coordination exerts strong restriction on the motions of loop L10 relative to loops L3 and L4. Principal component (PC) analysis also demonstrates that zinc ion binding totally inhibits the motion extent of NDM-1 and changes internal motion modes in NDM-1. We expect that the current study can provide significant dynamical information involving conformational changes of NDM-1 for the development of efficient inhibitors to decrease drug resistance of NDM-1 toward antibiotics.

Mechanics of torque generation in the bacterial flagellar motor

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual "power stroke." Specifically, we propose that ion-induced conformational changes about a proline "hinge" residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque-speed and speed-ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

Ca 2 + and Mg 2 + binding induce conformational stability of Calfumirin-1 from Dictyostelium discoideum

The apo-Calfumirin-1 (CAF-1) binds to Ca2+ with high affinity and also to Mg2+ with high positive cooperativity. The thermal unfolding curves of wtCAF-1 monitored at neutral pH by CD spectroscopy are reversible and show different thermal stabilities in the absence or presence of Ca2+ and Mg2+ ions. Metal-free wtCAF-1 shows greater thermal stability than EF-IV mutant protein. We observed that GdnHCl-induced unfolding of apo-wtCAF-1 monitored by CD and fluorescence spectroscopies increases co-operative folding with approximately same Cm values. Binding of Ca2+ and Mg2+ ions to CAF-1 dramatically altered the fluorescence and CD spectra, indicating metal ion-induced conformational changes both in the wild-type and mutant proteins. The hydrophobic probe, ANS is used to observe alteration in surface hydrophobicity of the protein in different ligation states. In apo-wtCAF-1, the exposed hydrophobic surfaces are able to bind ANS which is in contrast to the unfolded or the metal ions ligated conformations. Isothermal titration calorimetry (ITC) results show two possible independent binding sites of comparable affinity for the metal ions. However, their binding to the EF-IV E helix-loop-F helix mutant apo-protein happens with different affinities. The present study demonstrates that Ca2+ or Mg2+ binding plays a possible role in the conformational stability of the protein. This study aims to understand the mechanisms of Ca2+ and Mg2+ binding in CAF-1 using biophysical methods and correlate its improvement in D. discoideum cell differentiation. Results clearly indicate that wtCAF-1 is more stable than the mutant and retains its activity in Ca2+ bound form.

Protein Sizing and Conformation Analysis with an Electro-Switchable DNA Chip

Powerful methods for analyzing proteins and their interactions with small molecules, nucleic acids or other proteins are a cornerstone in the development of new drugs and next-generation disease diagnostics. Commonly, affinity and kinetics of molecular interactions are determined with surface biosensors, which measure the adsorption/desorption of solute target molecules to/from surface immobilized capture molecules. However, information on protein size, shape (folding state), molecular aggregates, or chemically subtle changes such as post-translational modifications, usually remain unrevealed and must be identified in complementary assays.Here, we show that by analyzing the molecular dynamics of proteins, set in motion by electrically actuated DNA ‘levers’, it is possible to measure protein size and detect changes in protein structure. Simultaneously, chemical rate constants and dissociation constants are obtained. The method is label-free, uses a parallel microelectrode format for multiplexed assays and microfluidics for low sample consumption. An analytical model based on continuum electrostatic Poisson-Boltzmann theory and the Fokker-Planck equation allows to determine the protein's diameter with sub-nanometer accuracy. Evaluating an extensive set of molecular dynamics data with ligand proteins of differing size, we find excellent agreement between theory and experiment.The devised concepts open up a novel route for protein analysis on a chip. The relative abundance of antibody fragments in heterogeneous mixtures can be determined, the unfolding of the protein tertiary structure is easily detected and even the ion-induced conformational change of a protein is observed in real-time. Moreover, we demonstrate the revelation of post-translational modifications at the example of a glycosylation and a phosphorylation.Due to the unprecedented information content about the investigated proteins, offered by this novel technology, we believe that we can provide an essential prerequisite for the progress in early identification of promising leads when screening for biological drug candidates.

Global Structure of Forked DNA in Solution Revealed by High-Resolution Single-Molecule FRET

Branched DNA structures play critical roles in DNA replication, repair, and recombination in addition to being key building blocks for DNA nanotechnology. Here we combine single-molecule multiparameter fluorescence detection and molecular dynamics simulations to give a general approach to global structure determination of branched DNA in solution. We reveal an open, planar structure of a forked DNA molecule with three duplex arms and demonstrate an ion-induced conformational change. This structure will serve as a benchmark for DNA-protein interaction studies.

Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States

NhaA, the main sodium–proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H and Na. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na and Li at pH above 7 and involves a 7-Å displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.

Exploring the Dynamics of Calix[4]pyrrole: Effect of Solvent and Fluorine Substitution

Molecular dynamics simulations show that calix[4]pyrrole (CP) and octafluorocalix[4]pyrrole (8F-CP) are extremely flexible molecules. CP mainly adopts the 1,3-alternate conformation in all the solvents, although the percentage of alternative conformations increases in polar solvents, especially those with good hydrogen-bonding acceptor properties. However, in the case of 8F-CP, the cone conformation is the most populated in some solvents. Transitions between conformers are common and fast, and both CP and 8F-CP can adopt the cone conformation needed for optimum interaction with anions more easily than would be predicted on the basis of previous gas-phase calculations. Furthermore, the present studies show that when a fluoride anion is specifically placed initially in close proximity to CP and 8F-CP in their respective 1,3-alternate conformations, an extremely fast change to the cone conformation is observed in both cases. The results suggest that preorganization does not represent a major impediment to anion-binding for either CP or 8F-CP, and that ion-induced conformational changes can follow different mechanisms depending on the solvent and the chemical substituents present on the calix[4]pyrrole beta-pyrrolic positions.

Selective and Sensitive Fluorescent Sensors for Metal Ions Based on Manipulation of Side-Chain Compositions of Poly(p-phenyleneethynylene)s

The syntheses and metal-responsive properties of poly(p-phenyleenethynylene)s with grafted new pseudo-crown-ether groups are reported. These polymers exhibit high sensitivities to alkali ions because of their collective optical properties, which are very sensitive to ion-induced conformational changes. The quenching of polymer fluorescence caused by the conformational changes is proportional to the ion concentration. The selectivity of the sensing materials toward Li(+) ions is significantly enhanced by controlling the size of the binding site via manipulation of the polymer side-chain compositions. The polymers are very stable for their six-month solid-state storage at room temperature.

Characterization of the Metal Ion Binding Properties of the Hepatitis C Virus RNA Polymerase

The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg(2+) or Mn(2+) ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K(d) values for the binding of Mg(2+) and Mn(2+) ions to the free enzyme were 3.1 and 0.3 mm, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ion-induced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state.