Charged Binding Sites Research Articles

Removal of Arsenate from Aqueous Solution by Adsorption onto Titanium Dioxide Nanoparticles

Titanium dioxide (TiO2) was investigated for the removal of pentavalent arsenate from aqueous solution. Kinetic results revealed that arsenate adsorption was almost instantaneous. The extent of arsenate adsorption decreased with increasing pH owing to the decrease of positively charged binding sites on the TiO2 surface. Adsorption isotherms measured at pH 3 and 7 generally followed the Langmuir model. The maximum uptake capacity ranged from 8 mg g−1 at pH 3 to 2.7 mg g−1 at pH 7. Addition of phosphate resulted in a significant reduction in arsenate adsorption, indicating that phosphate—a molecular analogue of arsenate—competes with arsenate for the same surface binding sites. By contrast, bicarbonate had little effect on arsenate adsorption, whereas sulfate exhibited a moderate suppression effect. A considerable reduction in arsenate adsorption was also observed in the presence of relatively high concentrations of background electrolytes (>50 mmol L−1).

Probing Molecular Docking in a Charged Model Binding Site

A model binding site was used to investigate charge-charge interactions in molecular docking. This simple site, a small (180A(3)) engineered cavity in cyctochrome c peroxidase (CCP), is negatively charged and completely buried from solvent, allowing us to explore the balance between electrostatic energy and ligand desolvation energy in a system where many of the common approximations in docking do not apply. A database with about 5300 molecules was docked into this cavity. Retrospective testing with known ligands and decoys showed that overall the balance between electrostatic interaction and desolvation energy was captured. More interesting were prospective docking scre"ens that looked for novel ligands, especially those that might reveal problems with the docking and energy methods. Based on screens of the 5300 compound database, both high-scoring and low-scoring molecules were acquired and tested for binding. Out of 16 new, high-scoring compounds tested, 15 were observed to bind. All of these were small heterocyclic cations. Binding constants were measured for a few of these, they ranged between 20microM and 60microM. Crystal structures were determined for ten of these ligands in complex with the protein. The observed ligand geometry corresponded closely to that predicted by docking. Several low-scoring alkyl amino cations were also tested and found to bind. The low docking score of these molecules owed to the relatively high charge density of the charged amino group and the corresponding high desolvation penalty. When the complex structures of those ligands were determined, a bound water molecule was observed interacting with the amino group and a backbone carbonyl group of the cavity. This water molecule mitigates the desolvation penalty and improves the interaction energy relative to that of the "naked" site used in the docking screen. Finally, six low-scoring neutral molecules were also tested, with a view to looking for false negative predictions. Whereas most of these did not bind, two did (phenol and 3-fluorocatechol). Crystal structures for these two ligands in complex with the cavity site suggest reasons for their binding. That these neutral molecules do, in fact bind, contradicts previous results in this site and, along with the alkyl amines, provides instructive false negatives that help identify weaknesses in our scoring functions. Several improvements of these are considered.

Enhanced adsorption of arsenate on titanium dioxide using Ca and Mg ions

In this study, we report the effects of pH and divalent cations on the adsorption of arsenate (As(V)) by titanium dioxide (TiO2) nanoparticles. The extent of As(V) adsorption on TiO2 decreased with increasing pH due to the decrease of positively charged binding sites on the TiO2 surface. The Langmuir maximum uptake capacity at pH 4 is about three times higher than that at pH 7. Here we show that the relatively low As(V) uptake at circumneutral pH could be substantially enhanced by the addition of common divalent cations such as magnesium and calcium. At a concentration of approximately 7 mM, magnesium and calcium increased the extent of As(V) adsorption from 2.1 to 6.5 and 7.7 mg As(V)/g TiO2, respectively.

Thorium Complexation by Hydroxamate Siderophores in Perturbed Multicomponent Systems Using Flow Injection Electrospray Ionization Mass Spectrometry

Flow injection electrospray ionization mass spectrometry has been shown to produce simple, characteristic m/z signals for Th-hydroxamate siderophore (desferrioxamine and ferrichrome) complexes, with Th complexed as a simple 4+ ion in the environmentally relevant pH range investigated (pH 5-9). All species of interest for this study were identified optimally in the positive mode; thus, multiple species were analyzed concurrently in a single spectrum. Complexation of Th by the two siderophores was rapid in 1:1 molar aqueous solution, reaching equilibrium before the first measurement was possible at 2 min. However, a significant proportion of the equimolar siderophore remained uncomplexed. Both siderophores rapidly exchanged Th for Fe when equimolar Fe(III) was added to the Th complexes, and only a small proportion of each siderophore remained complexed with Th at equilibrium (7-30 min). The results show a difference in the affinities of the two siderophores for the metals; ferrichrome has a 5-fold higher affinity than desferrioxamine for Th and a 5-fold lower affinity than desferrioxamine for Fe. Also, siderophore-complexed Th interacted strongly with a cation-exchange resin suggesting that, even when complexed by trianionic siderophores, Th mobility will be impeded by interactions with negatively charged binding sites in subsurface environmental matrixes. These results have important implications regarding siderophore-enhanced actinide(IV) mobility in the terrestrial environment.

Virtual Screening against Highly Charged Active Sites: Identifying Substrates of Alpha−Beta Barrel Enzymes

We have developed a virtual ligand screening method designed to help assign enzymatic function for alpha-beta barrel proteins. We dock a library of approximately 19,000 known metabolites against the active site and attempt to identify the relevant substrate based on predicted relative binding free energies. These energies are computed using a physics-based energy function based on an all-atom force field (OPLS-AA) and a generalized Born implicit solvent model. We evaluate the ability of this method to identify the known substrates of several members of the enolase superfamily of enzymes, including both holo and apo structures (11 total). The active sites of these enzymes contain numerous charged groups (lysines, carboxylates, histidines, and one or more metal ions) and thus provide a challenge for most docking scoring functions, which treat electrostatics and solvation in a highly approximate manner. Using the physics-based scoring procedure, the known substrate is ranked within the top 6% of the database in all cases, and in 8 of 11 cases, it is ranked within the top 1%. Moreover, the top-ranked ligands are strongly enriched in compounds with high chemical similarity to the substrate (e.g., different substitution patterns on a similar scaffold). These results suggest that our method can be used, in conjunction with other information including genomic context and known metabolic pathways, to suggest possible substrates or classes of substrates for experimental testing. More broadly, the physics-based scoring method performs well on highly charged binding sites and is likely to be useful in inhibitor docking against polar binding sites as well. The method is fast (<1 min per ligand), due largely to an efficient minimization algorithm based on the truncated Newton method, and thus, it can be applied to thousands of ligands within a few hours on a small Linux cluster.

Mutational Analysis of the Complement Receptor Type 2 (CR2/CD21)–C3d Interaction Reveals a Putative Charged SCR1 Binding Site for C3d

We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.

Lanthanum Effect on the Dynamics of Tight Junction Opening and Closing

We present a comparative study in frog urinary bladders (FUB) and A6 cell monolayers (A6CM) on the effect of La3+ on tight junction (TJ) dynamics. These tissues react similarly to changes of basolateral Ca2+ (Ca(2+)bl), while responding differently to the action of La3+(bl). In FUB, La(3+)bl shows a Ca(2+)-antagonistic effect that promotes TJ opening in the presence of a normal Ca(2+)bl concentration. In A6CM, in contrast, La(3+)bl always shows a clear Ca(2+)-agonistic effect. The fact that a concentration of La(3+)bl one fifth of the normal Ca(2+)bl leads in FUB to TJ opening and in A6CM to a complete recovery of the TJ seal indicates a high affinity of La3+ for the Ca(2+)-binding sites in both tissues. In FUB, apical La3+ (La(3+)ap) exhibits, differently from its basolateral effect, an evident Ca(2+)-agonistic effect, suggesting a dual effect of La3+, depending on which side of the bladder La3+ is applied. In A6CM La(3+)ap has a Ca(2+)-agonistic effect similar to La(3+)bl. The effects of La(3+)bl in FUB and in A6CM are consistent, according to our previous publications, with La3+ acting antagonistically or agonistically, respectively, on the Ca2+ binding sites of zonula adhaerens. Despite the fact that the effect of La(3+)ap is clear in both tissues, its site of action is yet to be determined. Protonation of the Ca(2+)-binding sites causes a decrease of its agonistic effect on A6CM, consistent with a negatively charged binding site. In A6CM La3+ apparently replaces Ca2+, mimicking the effect of Ca2+ triggering the cascade of events leading to TJ closure. In FUB, La3+ interacts with the binding sites, dislodging Ca2+, with a high affinity, but this interaction is inadequate to initiate or sustain the process of junction closing. Possibly, the difference between the two preparations resides in subtle conformation differences of the outer segment of E-cadherin molecules.

P-glycoprotein-mediated multidrug resistance phenotype of L1210/VCR cells is associated with decreases of oligo- and/or polysaccharide contents.

Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.

Essential role of conserved arginine 160 in intramolecular electron transfer in human sulfite oxidase.

Arginine 160 in human sulfite oxidase (SO) is conserved in all SO species sequenced to date. Previous steady-state kinetic studies of the R160Q human SO mutant showed a remarkable decrease in k(cat)/K(m)(sulfite) of nearly 1000-fold, which suggests that Arg 160 in human SO makes an important contribution to the binding of sulfite near the molybdenum cofactor [Garrett, R. M., Johnson, J. L., Graf, T. N., Feigenbaum, A., Rajagopalan, K. V. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6394-6398]. In the crystal structure of chicken SO, Arg 138, the equivalent of Arg 160 in human SO, is involved in the formation of a positively charged sulfite binding site [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., Rees, D. C. (1997) Cell 91, 973-983]. To further assess the role of Arg 160 in human SO, intramolecular electron transfer (IET) rates between the reduced heme [Fe(II)] and oxidized molybdenum [Mo(VI)] centers in the wild type, R160Q, and R160K human SO forms were investigated by laser flash photolysis. In the R160Q mutant, the IET rate constant at pH 6.0 was decreased by nearly 3 orders of magnitude relative to wild type, which indicates that the positive charge of Arg 160 is essential for efficient IET in human SO. Furthermore, the IET rate constant for the R160K mutant is about one-fourth that of the wild type enzyme, which strongly indicates that it is the loss of charge of Arg 160, and not its precise location, that is responsible for the much larger decrease in IET rates in the R160Q mutant. Steady-state kinetic measurements indicate that IET is rate-limiting in the catalytic cycle of the R160Q mutant. Thus, the large decrease in the IET rate constant rationalizes the fatal impact of this mutation in patients with this genetic disorder.

Antithrombin III Phenylalanines 122 and 121 Contribute to Its High Affinity for Heparin and Its Conformational Activation

The dissociation equilibrium constant for heparin binding to antithrombin III (ATIII) is a measure of the cofactor's binding to and activation of the proteinase inhibitor, and its salt dependence indicates that ionic and non-ionic interactions contribute approximately 40 and approximately 60% of the binding free energy, respectively. We now report that phenylalanines 121 and 122 (Phe-121 and Phe-122) together contribute 43% of the total binding free energy and 77% of the energy of non-ionic binding interactions. The large contribution of these hydrophobic residues to the binding energy is mediated not by direct interactions with heparin, but indirectly, through contacts between their phenyl rings and the non-polar stems of positively charged heparin binding residues, whose terminal amino and guanidinium groups are thereby organized to form extensive and specific ionic and non-ionic contacts with the pentasaccharide. Investigation of the kinetics of heparin binding demonstrated that Phe-122 is critical for promoting a normal rate of conformational change and stabilizing AT*H, the high affinity-activated binary complex. Kinetic and structural considerations suggest that Phe-122 and Lys-114 act cooperatively through non-ionic interactions to promote P-helix formation and ATIII binding to the pentasaccharide. In summary, although hydrophobic residues Phe-122 and Phe-121 make minimal contact with the pentasaccharide, they play a critical role in heparin binding and activation of antithrombin by coordinating the P-helix-mediated conformational change and organizing an extensive network of ionic and non-ionic interactions between positively charged heparin binding site residues and the cofactor.

Molecular Determinants of Permeation through the Cation Channel TRPV4

We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.

Parameter optimized surfaces (POPS): analysis of key interactions and conformational changes in the ribosome.

We present a new method for the calculation of solvent accessible surface areas at the atomic and residue levels, which we call parameter optimized surfaces (POPS-A and POPS-R ). Atomic and residue areas (the latter simulated with a single sphere centered at the C(alpha)s atom for amino acids and at the P atom for nucleotides) have been optimized versus accurate all-atoms methods. We concentrated on an analytical formula for the approximation of solvent accessibilities. The formula is simple, easily derivable and fast to compute, therefore it is practical for use in molecular dynamics simulations as an approximation to the first solvation shell. The residue based approach POPS-R has been derived as a useful tool for the analysis of large macromolecular assemblies like the ribosome, and is especially suited for use in refinement of low resolution structures. The structures of the 70S, 50S and 30S ribosomes have been analyzed in detail and most of the interactions within the subunits and at their interfaces were clearly identified. Some interesting differences between 30S alone and within the 70S have been highlighted. Owing to the presence of the P-tRNA in the 70S ribosome, localized conformational rearrangements occur within the subunits, exposing Arg and Lys residues to negatively charged binding sites of P-tRNA. POPS-R also allows for estimates of the loss of free energy of solvation upon complex formation, particularly useful in designing new protein-RNA complexes and in suggesting more focused experimental work.

Conformation and stability of alpha-helical membrane proteins. 1. Influence of salts on conformational equilibria between active and Inactive states of rhodopsin.

We studied the influence of salts on the pH-dependent conformational equilibria between the active and the inactive photoproduct states of rhodopsin, Meta II and Meta I, respectively, and between the active and inactive conformations of the apoprotein opsin. In both equilibria, the active species is favored in the presence of medium to high concentration of salt. The ion selectivity for the Meta I/Meta II equilibrium is particularly pronounced for the anions and follows the series trichloroacetate > thiocyanate > iodide > bromide > sulfate > chloride > acetate. The Hill coefficient of this salt-induced transition is close to 2.0. Both ion selectivity and Hill coefficient suggest that the transition is mainly regulated by ion binding to two specific charged binding sites in the protein with smaller contributions being due to the Hofmeister effect. We propose that these putative ion binding sites are identical to those sites that are titrated in the corresponding pH-dependent conformational transition. They presumably function as ionic locks, which keep the receptor in an inactive conformation, and which may be disrupted either by pH-dependent protonation or by salt-dependent ion binding.

The disulfonic stilbene DIDS and the marine poison maitotoxin activate the same two types of endogenous cation conductance in the cell membrane of Xenopus laevis oocytes.

In the present experiments we exposed the intra- or extracellular surface of excised giant membrane patches of Xenopus laevis oocytes bathed in 140 mmol/l Na-aspartate solution to the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS, 250 micromol/l). We observed that DIDS activated at least two cation conductances: (1) a non-selective cation (NSC) conductance that was mediated by channels of approximately 27 pS and resembled the stretch-activated cation conductance that has been observed in the oocyte cell membrane previously, and (2) a Na+-selective conductance, the single-channel events of which could not be resolved and which resembled the depolarization-induced Na+ conductance that has also been observed in the oocyte cell membrane previously. Both conductances were blocked by 1 mmol/l amiloride from the intra- and extracellular surfaces but inhibition of the NSC conductance by extracellular amiloride was less pronounced. Both conductances activated only slowly with a delay of 15-60 s after application of DIDS and remained active even after DIDS was washed off. This suggests that DIDS caused the exocytosis of preformed channels and this interpretation was supported by our additional observation that extracellular application of maitotoxin (MTX) mimicked the effects of DIDS. MTX is a marine toxin that has recently been reported to induce exocytosis in Xenopus laevis oocytes. The fact that DIDS and MTX each carry two sulfonyl groups suggests that they act on the same positively charged binding sites of an exocytosis-inducing protein. Our observations demonstrate that using DIDS to inhibit heterologously expressed anion transporters in the cell membrane of Xenopus laevis oocytes may compromise proper determination of the transporter currents. This effect can be prevented if the DIDS-activated endogenous cation conductances are suppressed by application of amiloride to the cytoplasmic surface of the cell membrane.

A theoretical study of electronic and structural states of neurotransmitters: gamma-aminobutyric acid and glutamic acid.

As a first approach to understanding the mechanism for the recognition of a ligand by its receptor, we first calculated the electronic and structural states of ionized gamma-aminobutyric acid (GABA) and ionized glutamic acid using the ab initio method with the 6-311++G (3df, 2pd) basis set. We paid special attention to the physicochemical characteristics of these molecules, such as the electric dipole moment, electrostatic potential, and electrostatic force. Even though GABA and glutamic acid are known to exert completely opposite influences in the mammalian brain by binding their specific receptors, the only difference in their chemical structures is that glutamic acid contains one more carboxyl group than GABA. As a result, we succeeded in showing that a difference of only one carboxyl group induces significant differences in the electronic and structural states between these molecules. These differences have a crucial influence on the electric dipole moments, the electrostatic potentials, and the electrostatic forces. The most remarkable finding of the present research is that the electrostatic potential formed by glutamic acid is composed of only negative parts, while that formed by GABA is separated into positive and negative parts. These results strongly suggest that GABA can approach either positively or negatively charged amino acids by adjusting its own orientation, while glutamic acid can approach only a positively charged binding site.

The toxicity of the antiparasitic mixture FMC is changed by humic substances and calcium

An inexpensive mixture of the therapeutic agents malachite green (MAG), methylene blue (MB) and formalin (known as FMC) is used to control external fungal infections in fish eggs (e.g. Saprolegnia ssp., Achlya ssp.) in aquaculture and ornamental fish breeding. The treatment success as well as the toxicity of the chemicals is determined by the toxicity of each of the substances and synergistic effects among them. Moreover, toxicity and therapeutical index might be affected by water parameters such as the ion content and the presence of humic substances (HS). Four different test waters with combinations of low (0.2 mmol L−1) and high (2 mmol L−1) calcium (Ca), +HS (5 mg L−1 C) or –HS (0 mg L−1 C) were used to determine the toxicity of FMC to early life stages of zebrafish, Danio rerio (Hamilton-Buchanan, 1822, 1823). Thus, the test solutions differed only in the content of dissolved organic carbon, and/or the amount of Ca chloride, and, to a lesser extent, pH. Zebrafish eggs reaching the four- to eight-cell stage were exposed to different concentrations of FMC (10, 15, 22, 33, 50 µL L−1 FMC) for 144 h. Survival was highest in low Ca/+HS-solution. High Ca and HS did not better protect the eggs than HS alone, so that this group had the lowest survival. Survival of the ‘–HS-groups’ was intermediate. The toxicity of the mixture as well as the interaction with Ca and HS was mainly determined by MAG. MAG, the most toxic part of the mixture, is positively charged and competes with Ca for the negatively charged binding sites of the HS. In presence of high Ca, the binding sites of the HS are filled with Ca-ions. This causes lower binding of MAG to the HS. Thus, MAG binds to the juvenile fish and enhances the toxicity of the mixture. Therefore, the toxicity as well as the therapeutical index may be altered in the presence of HS and the Ca content of the water.

Role of the N-terminal region of ribosomal protein S7 in its interaction with 16S rRNA which binds to the concavity formed by the beta-ribbon arm and the alpha-helix.

The ribosomal protein S7, a primary 16S rRNA-binding protein, plays an essential role in stabilizing the 3' major domain of 16S rRNA and also in feedback regulation of the str operon, as a translational repressor. We examined amino acid residues in ribosomal protein S7 from Bacillus stearothermophilus (BstS7) that are essential for 16S rRNA binding. Truncation of the N-terminal 10 residues of BstS7 abolished its binding to 16S rRNA, whereas removal of the C-terminal eight residues had no effect on the binding activity. Subsequently, we used site-directed mutagenesis to identify essential basic residues in the N-terminal region for 16S rRNA binding. Mutation of Arg3 and Lys8 significantly weakened the binding activity, and a smaller decrease in binding activity was observed with Arg2 and Arg9 mutations. These observations indicate that N-terminal basic residues, especially Arg3 and Lys8, play a crucial role as positively charged recognition groups for the negatively charged phosphate backbone of 16S rRNA. In addition, the mutagenesis study showed that Arg75, Arg78, Arg94, and Arg101, which are located in a concavity formed by the beta-ribbon arm and the alpha-helix (alpha4), individually make only a small contribution to 16S rRNA binding, but together probably form a positively charged binding site for 16S rRNA. With regard to aromatic residues, Tyr84 on the tip of the beta-ribbon arm was found to be involved in 16S rRNA binding, whereas the conserved aromatic residues Trp102 and Tyr106 in the concavity had little effect. We then probed the 16S rRNA-binding site(s) for the N-terminal region of S7 with iron tethered to the mutant of BstS7 containing a single cysteine residue at position 4. The N-terminal region of S7 is placed in close proximity to helix 43 in the 16S rRNA. Probing also revealed additional cleavages between nucleotides 1397 and 1438, near the P-site region in 16S rRNA. This finding is consistent with a three-dimensional model of 16S rRNA that shows close proximity of helix 43 to the P-site during three-dimensional folding.

Nature of the Chromophore Binding Site of Bacteriorhodopsin: The Potential Role of Arg 82 as a Principal Counterion

The nature of the chromophore binding site of light-adapted bacteriorhodopsin is analyzed by using modified neglect of differential overlap with partial single and double configuration interaction (MNDO-PSDCI) molecular orbital theory to interpret previously reported linear and nonlinear optical spectroscopic measurements. We conclude that in the absence of divalent metal cations in close interaction with Asp 85 and Asp 212, a positively charged amino acid must be present in the same vicinity. We find that models in which Arg 82 is pointed upward into the chromophore binding site and directly stabilizes Asp 85 and Asp 212 are successful in rationalizing the observed one-photon and two-photon properties. We conclude further that a water molecule is strongly hydrogen bonded to the chromophore imine proton. The chromophore “ 1B u* +” and “ 1A g* −” states, despite extensive mixing, exhibit significantly different configurational character. The lowest-lying “ 1B u* +” state is dominated by single excitations, whereas the second-excited “ 1A g* −” state is dominated by double excitations. We can rule out the possibility of a negatively charged binding site, because such a site would produce a lowest-lying “ 1A g* −” state, which is contrary to experimental observation. The possibility that Arg 82 migrates toward the extracellular surface during the photocycle is examined.

Matrix and modifier effects in the supercritical fluid extraction of cocaine and benzoylecgonine from human hair.

The supercritical fluid extraction (SFE) behavior of cocaine and its major metabolite benzoylecgonine (BZE) was investigated and found to be highly dependent upon the chemical nature of the matrix and the manner in which the target drug analytes are incorporated into or on the matrix. The recovery of cocaine from Teflon wool, filter paper, drug-fortified hair, and drug user hair was studied using a variety of CO2/modifier mixtures. Incorporation of a triethylamine (TEA)/water modifier mixture provided dramatic improvements in the recovery of cocaine from interactive matrixes. The results suggest that the SF extractability of cocaine is not limited by analyte solubility; rather, desorption of cocaine from hair binding sites is a rate-limiting step in the SFE process. A displacement SFE mechanism is hypothesized in which TEA (as the triethylammonium cation) competes with cocaine for negatively charged hair binding sites. The dependence of extractability on hair/drug binding interactions allows the differentiation of cocaine present at different discrete sites in hair based on differences in SFE behavior. These findings suggest the potential for distinguishing exogenous (i.e., environmental) from endogenous (i.e., physiological) sources of drugs in hair. In contrast to the results observed for cocaine, SFE recoveries of BZE were poor from all matrixes and under all conditions studied. Its increased polarity, the presence of an additional binding site, and the possibility of multiple charged states suggest that poor BZE recoveries may be due to both poor analyte solubility and failure to desorb the analyte from hair binding sites under the conditions employed.

Electron transfer reactions of Anabaena PCC 7119 ferredoxin:NADP + reductase with nonphysiological oxidants

The mechanism of single-electron oxidation of ferredoxin-NADP + reductase (FNR) (EC 1.18.1.2) from cyanobacterium Anabaena PCC 7119 by quinones, aromatic nitrocompounds and inorganic complexes has been studied. In steady-state experiments, the logarithms of bimolecular rate constants of reduction of quinones and nitroaromatics increase with an increase in their single-electron reduction potential, the reactivities of nitroaromatics being markedly lower than of quinones. The absence of inhibition of reaction by ferredoxin and insignificant ionic strength effects suggest that positively charged ferredoxin binding site of FNR is not involved in reduction. In stopped-flow kinetics of oxidation of photoreduced enzyme by 5,8-dihydroxy-1,4-naphthoquinone, the oxidation of FADH . to FAD proceeds much slower than oxidation of FADH - to semiquinone. The patterns of reaction inhibition by NADP + and 2′,5′-ADP also suggest that oxidation of FAD semiquinone is a rate-limiting step in oxidative half-reaction of steady-state experiments. The analysis of reaction kinetics within the framework of `outer-sphere' electron transfer model gives the values of electron self-exchange constants of FAD/FADH . couple and site-to-surface distances, 0.82–0.95 nm, that seem overestimated in view of available data on the accessibility of FAD to solvent. A possible explanation of poor reactivity of FAD/FADH . redox couple of ferredoxin:NADP + reductase in comparison to FADH ./FADH − couple is that oxidation of FADH − to semiquinone re-presents a `pure' electron transfer, whereas oxidation of FADH . to FAD is electron transfer coupled to a slower proton transfer.