Ionizable Side Chains Research Articles

Revelation of a catalytic calcium-binding site elucidates unusual metal dependence of a human apyrase.

Human soluble calcium-activated nucleotidase 1 (hSCAN-1) represents a new family of apyrase enzymes that catalyze the hydrolysis of nucleotide di- and triphosphates, thereby modulating extracellular purinergic and pyrimidinergic signaling. Among well-characterized phosphoryl transfer enzymes, hSCAN-1 is unique not only in its unusual calcium-dependent activation, but also in its novel phosphate-binding motif. Its catalytic site does not utilize backbone amide groups to bind phosphate, as in the common P-loop, but contains a large cluster of acidic ionizable side chains. By employing a state-of-the-art computational approach, we have revealed a previously uncharacterized catalytic calcium-binding site in hSCAN-1, which elucidates the unusual calcium-dependence of its apyrase activity. In a high-order coordination shell, the newly identified calcium ion organizes the active site residues to mediate nucleotide binding, to orient the nucleophilic water, and to facilitate the phosphoryl transfer reaction. From ab initio QM/MM molecular dynamics simulations with umbrella sampling, we have characterized a reverse protonation catalytic mechanism for hSCAN-1 and determined its free energy reaction profile. Our results are consistent with available experimental studies and provide new detailed insight into the structure-function relationship of this novel calcium-activated phosphoryl transfer enzyme.

Functional Conducting Polymers via Thiol-ene Chemistry.

We demonstrate here that thiol-ene chemistry can be used to provide side-chain functionalized monomers based on 3,4-propylenedioxythiophene (ProDOT) containing ionic, neutral, hydrophobic, and hydrophilic side chains. All reactions gave high yields and purification could generally be accomplished through precipitation. These monomers were polymerized either chemically or electro-chemically to give soluble materials or conductive films, respectively. This strategy provides for facile tuning of the solubility, film surface chemistry, and film morphology of this class of conducting polymers.

Structural studies of amphiphilic oligopeptides composed of alternating alanine and ionizable amino-acid residues using CD and 13C CP/MAS NMR spectroscopy

We report a novel self-assembling system of amphiphilic oligopeptides composed of alternating hydrophobic and ionizable amino-acid residues. Three types of peptides, AD12, AE12 and AK12, were prepared as building blocks for this model system. These peptides formed helical conformations in low-concentration solutions, but these peptides adopted β-sheet structures and precipitated in highly concentrated solutions at a pH that neutralizes the charges on the ionizable side chains. In addition, the mixing of the peptides with positive and negative charges on the side chains of the ionizable amino-acid residues was found to trigger the formation of the β-sheet structure. This system could be used to create new materials based on oligopeptides.

Enhancement of α-Helix Mimicry by an α/β-Peptide Foldamer via Incorporation of a Dense Ionic Side-Chain Array

We report a new method for preorganization of α/β-peptide helices, based on the use of a dense array of acidic and basic side chains. Previously we have used cyclically constrained β residues to promote α/β-peptide helicity; here we show that an engineered ion pair array can be comparably effective, as indicated by mimicry of the CHR domain of HIV protein gp41. The new design is effective in biochemical and cell-based infectivity assays; however, the resulting α/β-peptide is susceptible to proteolysis. This susceptibility was addressed via introduction of a few cyclic β residues near the cleavage site, to produce the most stable, effective α/β-peptide gp41 CHR analogue identified. Crystal structures of an α- and α/β-peptide (each involved in a gp41-mimetic helix bundle) that contain the dense acid/base residue array manifest disorder in the ionic side chains, but there is little side-chain disorder in analogous α- and α/β-peptide structures with a sparser ionic side-chain array. These observations suggest that dense arrays of complementary acidic and basic residues can provide conformational stabilization via Coulombic attractions that do not require entropically costly ordering of side chains.

Diffusion and binding of 5-fluorouracil in non-ionic hydrogels with interpolymer complexation

Hydrogen-bonded interpolymer complexes can be used for development of novel dosage forms. In this study, two types of crosslinked hydrogels, copolymer networks of N-vinyl pyrrolidone and acrylamide (PVP-co-PAM) and interpenetrating polymer networks (IPN) composed of crosslinked PVP-co-PAM and poly(vinyl alcohol) (PVA), were synthesized at three different degrees of crosslinking. The side chain groups in such polymers can form non-ionic complexes through H-bonding, resulting in additional “crosslinks” in the hydrogels. Both kinds of hydrogels have significantly larger swelling sensitivities than the networks formed with ionizable side chains. In the IPNs, introduction of the PVA chains into the PVP-co-PAM networks raises the permeability, indicating more open pores. The permeability decreases with the increasing degree of crosslinking of the copolymer. For probing the drug binding in the hydrogels, Fourier transform infrared spectra (FTIR) difference spectroscopy indicated the presence of significant H-bonding interactions between 5-fluorouracil (5-FU) and the side chains of the polymers. Such interactions are larger in the PVP-co-PAM copolymers than in the IPN hydrogels, thereby causing an additional source of the slower release kinetics in the copolymer hydrogels as revealed by the Peppas model, albeit both types of the networks followed a non-Fickian transport mechanism.

Ionizable side chains at catalytic active sites of enzymes

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072Å(3). The number density of acid side chains is 8.3M (in chemical units); the number density of basic side chains is 10.6M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

Quantum chemical modeling of rhodopsin mutants displaying switchable colors

We look at the possibility to compute and understand the color change occurring upon mutation of a photochromic protein. Accordingly, ab initio multiconfigurational quantum chemical methods are used to construct basic quantum-mechanics/molecular-mechanics (QM/MM) models for a small mutant library of the sensory rhodopsin of Anabaena (Nostoc) sp. PCC7120 cyanobacterium. Together with the wild-type forms, a set of 26 absorption maxima spanning a ca. 80 nm range is obtained. We show that these models can be used to capture the electrostatic change controlling the computed color variation and the change in the ionization of specific side chains.

The Ring of Glutamates in the Charge-Selectivity Filter Region of the Nicotinic Receptor Forms a System of Unanticipated Complexity

Of all the rings of ionizable side chains that decorate the permeation pathway of nicotinic-type receptors (AChRs), the ring of glutamates at the intracellular end of M2 (the “intermediate ring”) is, by far, the one that lowers the energetic cost of cation permeation the most. Although these glutamates have received much attention, several properties of this ring (such as the pH insensitivity) have remained unexplained, especially when compared to the properties of similar rings in CNG, Nav and Cav channels. To gain a more detailed understanding of this “catalytic” site, we performed a thorough mutational analysis of the intermediate ring of the muscle AChR (which consists of four glutamates and one glutamine) using single-channel electrophysiology. Our results indicate that the commonly held idea that each glutamate represents a negative charge and that each additional charge makes an incremental contribution to the single-channel conductance is incorrect: for example, we found that (in the wild-type channel) only two, not all four, of the glutamates contribute to the single-channel conductance. And, in mutant muscle AChRs bearing five glutamates in the ring (and thus mimicking the non-muscle type), only three glutamates (not five) contribute to the conductance. Our data point to a model of unanticipated complexity in which the conformational dynamics of the glutamate side chains (rather than their acid-base properties) in the ambivalent environment afforded by the membrane-water interface play a fundamental role in determining the amplitude of unitary currents.

Label-Free DNA Detection through Energy Transfer of Conjugated Polymer Complexes

Label-free DNA detection is a highly sensitive and selective method with great scientific and social significance. Water-soluble cationic conjugated polymer (CCP), poly(9,9-bis(6'-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene) (PFP), was synthesized by cross coupling reaction under the catalysis of palladium, and water-soluble anionic conjugated polymer (ACP), poly(3-thiophene sodium acetate) (P3TSA), was prepared through oxidative addition polymerization with anhydrous ferric chloride as the catalyst. Both PFP and P3TSA were well characterized by H nuclear magnetic resonance (H NMR) and Fourier transform infrared (FTIR) tests. Owing to the existence of opposite ionic side-chains, PFP and P3TSA were both water-soluble and could form polymer complexes through electrostatic interactions. The transmittance measurements of the polymer complex solution with wavelength ranging from 600 to 750 nm were performed. The results showed that the transmittance was almost unchanged for all samples, confirming the high solubility and stability of polymer complexes. The ultraviolet-visible (UV-vis) and photoluminescence emission measurements showed that the emission spectrum of PFP overlapped a lot with the absorption spectrum of P3TSA, proving the energy transfer from PFP (donor) to P3TSA (acceptor). By fixing the donor concentration, energy transfer efficiency (ETEF) of the polymer complexes increased with the concentration of P3TSA. With the enhancement of P3TSA concentration, the ETEF increased slowly at first and then relatively fast at a high P3TSA concentration. A sample with a high ETEF was selected to investigate the effect of DNA probe (X1) concentration on ETEF. It was observed that ETEF decreased with the increase of X1 concentration, owing to the repulsion of P3TSA by negative charged DNA probes. DNA hybridization was conducted by using 0.2 nmol of X1 as a probe. The experimental results showed that the perfectly matched, double mismatched and non-matched target DNA could be well distinguished with obvious photoluminescence signals. In conclusion, a new label-free DNA detection method with high selectivity has been successfully developed based on the energy transfer of conjugated polymer complexes.

Accuracy of Density Functionals in the Prediction of Electronic Proton Affinities of Amino Acid Side Chains.

The ionization states of amino acids influence the structure, function, stability, solubility, and reactivity of proteins and are difficult to determine unambiguously by experimental means. Thus, it is very important to be able to determine them theoretically and with high reliability. We have analyzed how well DFT functionals, often used to characterize complex and large models such as proteins, describe the zero-point-exclusive proton affinity at 0 K, PAel0K, for the ionizable side chains of lysine (Lys), histidine (His), arginine (Arg), and aspartate (Asp–) as well as the cysteine (Cys–), serine (Ser–), and tyrosine (Tyr–) anions. The reference values PAel0K were determined at the very accurate CCSD(T)/CBS level. Those values were obtained by the sum of the complete basis set limit of the MP2 energies plus a CCSD(T) correction term evaluated with the aug-cc-pVTZ basis set. The complete basis set limit of MP2 energies was determined using the Truhlar and Helgaker extrapolation schemes. A new, important,...

Interactions between Ionizable Amino Acid Side Chains at a Lipid Bilayer–Water Interface

Potentials of mean force (PMF) between ionizable amino acid side chains (Arg, Lys, His, Glu) in the headgroup area of a palmitoyl oleoyl phosphatidylcholine lipid bilayer were obtained from all-atom molecular dynamics simulations and the adaptive biasing force method. Simulations in bulk water were also performed for comparison. Side chains were constrained in collinear, stacking, and orthogonal (T-shaped) orientations. The most structured and attractive PMFs were observed for hydrogen-bonded side chains. Contact minima occurred at a distance of 2.6-3.1 Å between selected atoms or centers of mass with the most attractive interaction (-9.6 kcal/mol) observed between Arg(+) and Glu(-). Hydrogen bonds play a significant role in stabilizing these interactions. Interactions between like charged side chains can also be very attractive if the charges are screened by surrounding molecules or groups (e.g., the PMF value at the contact minimum for Arg(+)···Arg(+) is -7.6 kcal/mol). Like charged side chains can have contact minima as close as 3.6 Å. The PMFs depend strongly on the relative orientation of the side chains. In agreement with experimental studies and other simulations, we found the stacking arrangement of like charged side chains to be the most favorable orientation. Interaction energies and Lennard-Jones energies between side chains, headgroups, and water molecules were analyzed in order to rationalize the observed PMFs and their dependence on orientation. In general, the results cannot be explained by simple dielectric arguments.

Discrete M3-M4 Intracellular Loop Subdomains Control Specific Aspects of γ-Aminobutyric Acid Type A Receptor Function

The GABA type A receptor (GABA(A)R) is a member of the pentameric ligand gated ion channel (pLGIC) family that mediates ionotropic neurotransmission. Residues in the intracellular loop domain (ILD) have recently been shown to define part of the ion permeation pathway in several closely related members of the pentameric ligand gated ion channel family. In this study, we investigated the role the ILD of the GABA(A)R α1 subunit plays in channel function. Deletion of the α1 ILD resulted in a significant increase in GABA EC(50) and maximal current amplitude, suggesting that the ILD must be intact for proper receptor function. To test this hypothesis, we conducted a mutagenic screen of all amino acids harboring ionizable side chains within this domain to investigate the contribution of individual charged residues to ion permeation. Using macroscopic and single channel voltage-clamp recording techniques, we found that mutations within a subdomain of the α1 ILD near M3 altered GABA apparent affinity; interestingly, α1(K312E) exhibited reduced partial agonist efficacy. We introduced point mutations near M4, including α1(K383E) and α1(K384E), that enhanced receptor desensitization. Mutation of 5 charged residues within a 39-residue span contiguous with M4 reduced relative anion permeability of the channel and may represent a weak intracellular selectivity filter. Within this subdomain, the α1(K378E) mutation induced a significant reduction in single channel conductance, consistent with our hypothesis that the GABA(A)R α1 ILD contributes directly to the permeation pathway.

Influence of the membrane dipole potential on peptide binding to lipid bilayers

The implicit membrane model IMM1 is extended to include the membrane dipole potential and applied to molecular dynamics simulations of the helical peptides alamethicin, WALP23, influenza hemagglutinin fusion peptide, HIV fusion peptide, magainin, and the pre-sequence of cytochrome c oxidase subunit IV (p25). The results show that the orientation of the peptides in the membrane can be influenced by the dipole potential. The binding affinity of all peptides except for the hemagglutinin fusion peptide decreases upon increase of the dipole potential. The changes in both orientation and binding affinity are explained by the interaction of the dipole potential with the helix backbone dipole and ionic side-chains. In general, peptides that tend to insert the N-terminus in the membrane and/or have positively charged side chains will lose binding affinity upon increase of the dipole potential.

A possible CO2 conducting and concentrating mechanism in plant stomata SLAC1 channel.

Background The plant SLAC1 is a slow anion channel in the membrane of stomatal guard cells, which controls the turgor pressure in the aperture-defining guard cells, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought, high levels of carbon dioxide, and bacterial invasion. Recent study demonstrated that bicarbonate is a small-molecule activator of SLAC1. Higher CO2 and HCO3 – concentration activates S-type anion channel currents in wild-type Arabidopsis guard cells. Based on the SLAC1 structure a theoretical model is derived to illustrate the activation of bicarbonate to SLAC1 channel. Meanwhile a possible CO2 conducting and concentrating mechanism of the SLAC1 is proposed.MethodologyThe homology structure of Arabidopsis thaliana SLAC1 (AtSLAC1) provides the structural basis for study of the conducting and concentrating mechanism of carbon dioxide in SLAC1 channels. The pKa values of ionizable amino acid side chains in AtSLAC1 are calculated using software PROPKA3.0, and the concentration of CO2 and anion HCO3 – are computed based on the chemical equilibrium theory.ConclusionsThe AtSLAC1 is modeled as a five-region channel with different pH values. The top and bottom layers of channel are the alkaline residue-dominated regions, and in the middle of channel there is the acidic region surrounding acidic residues His332. The CO2 concentration is enhanced around 104 times by the pH difference between these regions, and CO2 is stored in the hydrophobic region, which is a CO2 pool. The pH driven CO2 conduction from outside to inside balances the back electromotive force and maintain the influx of anions (e.g. Cl– and NO3 –) from inside to outside. SLAC1 may be a pathway providing CO2 for photosynthesis in the guard cells.

Energetics of charge–charge interactions between residues adjacent in sequence

Statistical analysis of the residue separation between a pair of ionizable side chains within 4 Å of each other was performed on a set of 1560 non-homologous PDB structures. We found that the frequency of pairs of like charges (i.e., pairs consisting of acidic residues Asp and Glu or pairs consisting of basic residues Arg and Lys) is two orders of magnitude lower than the pairs of oppositely charged residues (salt-bridges). We also found that for pairs of like charges the distribution is skewed dramatically towards short residue separation (<3). On the basis of these observations, we hypothesize that at short residue separation the repulsion between charges does not contribute much to the protein stability and the effects are largely dominated by the long range charge-charge interactions with other ionizable groups in the protein molecule. To test this hypothesis, we incorporated various pairs of charged residues at position 63 and 64 of ubiquitin and compared the stabilities of these variants. We also performed calculations of the expected changes in the charge-charge interactions. A very good correlation between experimental changes in the stability of ubiquitin variants, and changes in the energy of charge-charge interactions provides support for the hypothesis that a pair of ionizable residues next to each other in sequence modulates protein stability via long range charge-charge interactions with the rest of the protein.

Estimating the pKa values of basic and acidic side chains in ion channels using electrophysiological recordings: A robust approach to an elusive problem

As a step toward gaining a better understanding of the physicochemical bases of pK(a)-value shifts in ion channels, we have previously proposed a method for estimating the proton affinities of systematically engineered ionizable side chains from the kinetic analysis of single-channel current recordings. We reported that the open-channel current flowing through mutants of the (cation-selective) muscle nicotinic acetylcholine receptor (AChR) engineered to bear single basic residues in the transmembrane portion of the pore domain fluctuates between two levels of conductance. Our observations were consistent with the idea that these fluctuations track directly the alternate protonation-deprotonation of basic side chains: protonation of the introduced basic group would attenuate the single-channel conductance, whereas its deprotonation would restore the wild-type-like level. Thus, analysis of the kinetics of these transitions was interpreted to yield the pK(a) values of the substituted side chains. However, other mechanisms can be postulated that would also be consistent with some of our findings but according to which the kinetic analysis of the fluctuations would not yield true pK(a)s. Such mechanisms include the pH-dependent interconversion between two conformations of the channel that, while both ion permeable, would support different cation-conduction rates. In this article, we present experimental evidence for the notion that the fluctuations of the open-channel current observed for the muscle AChR result from the electrostatic interaction between fixed charges and the passing cations rather than from a change in conformation. Hence, we conclude that bona fide pK(a) values can be obtained from single-channel recordings.

Tunable pKa values and the basis of opposite charge selectivities in nicotinic-type receptors.

Among ion channels, only the nicotinic-receptor superfamily has evolved to generate both cation- and anion-selective members. Although other, structurally unrelated, neurotransmitter-gated cation channels exist, no other type of neurotransmitter-gated anion channel, and thus no other source of fast synaptic inhibitory signals, has been described so far. In addition to the seemingly straightforward electrostatic effect of the presence (in the cation-selective members) or absence (in the anion-selective ones) of a ring of pore-facing carboxylates, mutational studies have identified other features of the amino-acid sequence near the intracellular end of the pore-lining transmembrane segments (M2) that are also required to achieve the high charge selectivity displayed by native channels1–10. However, the mechanism underlying this subtler effect has remained elusive11 and a subject of much speculation. Here, using single-channel electrophysiological recordings to estimate the protonation state of native ionizable side chains, we show that anion-selective type sequences favour, whereas cation-selective type sequences prevent, the protonation of the conserved, buried basic residues at the intracellular entrance of the pore (the M2 0′ position). We conclude that the, previously unrecognized, tunable charge state of the 0′ ring of buried basic side chains is an essential feature of these channels’ versatile charge-selectivity filter.

Conjugated polyelectrolytes for label-free visual detection of heparin

Conjugated polyelectrolytes (CPEs), with electron-delocalized backbones and charged ionic side chains, are unique sensory materials for the construction of optical biosensors. In this feature article, CPE-based visual detection of heparin is summarized. Three CPE-based heparin probes, including ratiometric, light-up and colormetric probes, are discussed in terms of molecular design, detection selectivity and quantification capability. The work summarized here provides fundamental guidelines for CPE-based label-free visual sensing.

Conformational relaxation and water penetration coupled to ionization of internal groups in proteins.

Molecular dynamics simulations were used to examine the effects of ionization of internal groups on the structures of eighteen variants of staphylococcal nuclease (SNase) with internal Lys, Asp, or Glu. In most cases the RMSD values of internal ionizable side chains were larger when the ionizable moieties were charged than when they were neutral. Calculations of solvent-accessible surface area showed that the internal ionizable side chains were buried in the protein interior when they were neutral and moved toward crevices and toward the protein-water interface when they were charged. The only exceptions are Lys-36, Lys-62, and Lys-103, which remained buried even after charging. With the exception of Lys-38, the number of internal water molecules surrounding the ionizable group increased upon charging: the average number of water oxygen atoms within the first hydration shell increased by 1.7 for Lys residues, by 5.2 for Asp residues, and by 3.2 for Glu residues. The polarity of the microenvironment of the ionizable group also increased when the groups were charged: the average number of polar atoms of any kind within the first hydration shell increased by 2.7 for Lys residues, by 4.8 for Asp residues, and by 4.0 for Glu residues. An unexpected correlation was observed between the absolute value of the shifts in pK(a) values measured experimentally, and several parameters of structural relaxation: the net difference in the polarity of the microenvironment of the charged and neutral forms of the ionizable groups, the net difference in hydration of the charged and neutral forms of the ionizable groups, and the difference in RMSD values of the charged and neutral forms of the ionizable groups. The effects of ionization of internal groups on the conformation of the backbone were noticeable but mostly small and localized to the area immediately next to the internal ionizable moiety. Some variants did exhibit local unfolding.

Anthraquinone‐oligodeoxynucleotide Conjugates as Triple Helix Stabilizing Agents

A number of anthraquinones have been synthesized and linked to a 10-mer homopyrimidine oligodeoxynucleotide. All the anthraquinone-oligodeoxynucleotide conjugates exhibited sequence specific triple helix formation when tested by DNase I footprinting. Neither the presence of an ionizable side chain nor the position of substitution of the anthraquinone appeared to affect triplex stability.