Multi‐cycle recovery of lactoferrin and lactoperoxidase from crude whey using fimbriated high‐capacity magnetic cation exchangers and a novel “rotor–stator” high‐gradient magnetic separator

Cerium (IV) initiated “graft‐from” polymerization reactions were employed to convert M‐PVA magnetic particles into polyacrylic acid‐fimbriated magnetic cation exchange supports displaying ultra‐high binding capacity for basic target proteins. The modifications, which were performed at 25 mg and 2.5 g scales, delivered maximum binding capacities (Q_max) for hen egg white lysozyme in excess of 320 mg g^?1, combined with sub‐micromolar dissociation constants (0.45–0.69 µm) and “tightness of binding” values greater than 49 L g^?1. Two batches of polyacrylic acid‐fimbriated magnetic cation exchangers were combined to form a 5 g pooled batch exhibiting Q_max values for lysozyme, lactoferrin, and lactoperoxidase of 404, 585, and 685 mg g^?1, respectively. These magnetic cation exchangers were subsequently employed together with a newly designed “rotor–stator” type HGMF rig, in five sequential cycles of recovery of lactoferrin and lactoperoxidase from 2 L batches of a crude sweet bovine whey feedstock. Lactoferrin purification performance was observed to remain relatively constant from one HGMF cycle to the next over the five operating cycles, with yields between 40% and 49% combined with purification and concentration factors of 37‐ to 46‐fold and 1.3‐ to 1.6‐fold, respectively. The far superior multi‐cycle HGMF performance seen here compared to that observed in our earlier studies can be directly attributed to the combined use of improved high capacity adsorbents and superior particle resuspension afforded by the new “rotor–stator” HGMS design. Biotechnol. Bioeng. 2013; 110: 1714–1725. © 2013 Wiley Periodicals, Inc.     

Novel N‐propylphthalimide‐ and 4‐vinylbenzyl‐substituted benzimidazole salts: Synthesis,characterization, and determination of their metal chelating effects and inhibition profiles against acetylcholinesterase and carbonic anhydrase enzymes

The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted N‐heterocyclic carbene (NHC) precursors were synthesized by N‐substituted benzimidazolium with aryl halides. The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted NHC precursors have been characterized by using ¹H NMR, ¹³C NMR, FTIR spectroscopy, and elemental analysis techniques. They were tested for the inhibition of AChE and hCA enzymes and demonstrated efficient inhibition profiles with K_i values in the range of 351.0–1269.9 nM against hCA I, 346.6–1193.1 nM against hCA II, and 19.0–76.3 nM against AChE. On the other hand, acetazolamide, a clinically used molecule, utilized as CA inhibitor, obtained a K_i value of 1246.7 nM against hCA I and 1407.6 nM against hCA II. Additionally, tacrine inhibited AChE and obtained a K_i value of 174.6 nM.     

Purification and Characterization of Glucose 6‐Phosphate Dehydrogenase, 6‐Phosphogluconate Dehydrogenase,and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide,Digoxin, and Dopamine on the Enzymes Activities

The present study was aimed to investigate characterization and purification of glucose–6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'‐ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS‐PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and K_i values with K_m and V_max values of the substrates and coenzymes were determined. According to the obtained K_i and IC₅₀ values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Temperature sensitivity of soil enzyme kinetics under N‐fertilization in two temperate forests

Soil microbes produce extracellular enzymes that degrade carbon (C)‐containing polymers in soil organic matter. Because extracellular enzyme activities may be sensitive to both increased nitrogen (N) and temperature change, we measured the effect of long‐term N addition and short‐term temperature variation on enzyme kinetics in soils from hardwood forests at Bear Brook, Maine, and Fernow Forest, West Virginia. We determined the V_max and K_m parameters for five hydrolytic enzymes: α‐glucosidase, β‐glucosidase, β‐xylosidase, cellobiohydrolase, and N‐acetyl‐glucosaminidase. Temperature sensitivities of V_max and K_m were assessed within soil samples subjected to a range of temperatures. We hypothesized that (1) N additions would cause microbial C limitation, leading to higher enzyme V_max values and lower K_m values; and (2) both V_max and K_m would increase at higher temperatures. Finally, we tested whether or not temperature sensitivity of enzyme kinetics is mediated by N addition. Nitrogen addition significantly or marginally significantly increased V_max values for all enzymes, particularly at Fernow. Nitrogen fertilization led to significantly lower K_m values for all enzymes at Bear Brook, but variable K_m responses at Fernow Forest. Both V_max and K_m were temperature sensitive, with Q₁₀ values ranging from 1.64–2.27 for enzyme V_max and 1.04–1.93 for enzyme K_m. No enzyme showed a significant interaction between N and temperature sensitivity for V_max, and only β‐xylosidase showed a significant interaction between N and temperature sensitivity for K_m. Our study is the first to experimentally demonstrate a positive relationship between K_m and temperature for soil enzymes. Higher temperature sensitivities for V_max relative to K_m imply that substrate degradation will increase with temperature. In addition, the V_max and K_m responses to N indicate greater substrate degradation under N addition. Our results suggest that increasing temperatures and N availability in forests of the northeastern US will lead to increased hydrolytic enzyme activity, despite the positive temperature sensitivity of K_m.     

The Michaelis–Menten kinetics of soil extracellular enzymes in response to temperature: a cross‐latitudinal study

Decomposition of soil organic matter (SOM) is mediated by microbial extracellular hydrolytic enzymes (EHEs). Thus, given the large amount of carbon (C) stored as SOM, it is imperative to understand how microbial EHEs will respond to global change (and warming in particular) to better predict the links between SOM and the global C cycle. Here, we measured the Michaelis–Menten kinetics [maximal rate of velocity (V_max) and half‐saturation constant (K_m)] of five hydrolytic enzymes involved in SOM degradation (cellobiohydrolase, β‐glucosidase, β‐xylosidase, α‐glucosidase, and N‐acetyl‐β‐d ‐glucosaminidase) in five sites spanning a boreal forest to a tropical rainforest. We tested the specific hypothesis that enzymes from higher latitudes would show greater temperature sensitivities than those from lower latitudes. We then used our data to parameterize a mathematical model to test the relative roles of V_max and K_m temperature sensitivities in SOM decomposition. We found that both V_max and K_m were temperature sensitive, with Q₁₀ values ranging from 1.53 to 2.27 for V_max and 0.90 to 1.57 for K_m. The Q₁₀ values for the K_m of the cellulose‐degrading enzyme β‐glucosidase showed a significant (P = 0.004) negative relationship with mean annual temperature, indicating that enzymes from cooler climates can indeed be more sensitive to temperature. Our model showed that K_m temperature sensitivity can offset SOM losses due to V_max temperature sensitivity, but the offset depends on the size of the SOM pool and the magnitude of V_max. Overall, our results suggest that there is a local adaptation of microbial EHE kinetics to temperature and that this should be taken into account when making predictions about the responses of C cycling to global change.     

Label‐free determination of protein–ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry

We performed a systematic comparison of three label‐free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. The performance of (1) nanoelectrospray ionization mass spectrometry (nESI‐MS), (2) surface plasmon resonance (SPR), and (3) isothermal titration calorimetry (ITC) was compared for the determination of dissociation constants (K_D). The model system studied for this purpose was the human carbonic anhydrase I (hCAI) with eight known and well characterized sulfonamide inhibitors (Krishnamurthy et al., Chem. Rev. 2008, 108: 946–1051). The binding affinities of the inhibitors chosen vary by more than four orders of magnitude e.g., the K_D value determined for ethoxzolamide by nESI‐MS was 5 ± 1 nM and the K_D value for sulfanilamide was 145.7 ± 10.0 µM. The agreement of the determined K_D values by the three methods investigated was excellent for ethoxzolamide and benzenesulfonamide (variation with experimental error), good for acetazolamide and 4‐carboxybenzenesulfonamide (variation by ～ one order of magnitude), but poor for others e.g., sulpiride. The accuracies of the K_D values are determined, and advantages and drawbacks of the individual methods are discussed. Moreover, we critically evaluate the three examined methods in terms of ease of the measurement, sample consumption, time requirement, and discuss their limitations. Copyright © 2009 John Wiley & Sons, Ltd.     

His‐tag binding by antibody C706 mimics β‐amyloid recognition

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β‐amyloid (Aβ) plaques. Aggregation of the Aβ₄₂ peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti‐Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2₁ and C2. Although the Fab was crystallized in the presence of Aβ₁₆, no peptide was observed in the crystals. The antigen‐binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly‐His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen‐binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his‐tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B‐cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.     

Desensitization of β‐adrenergic receptors in lung injury induced by 2‐chloroethyl ethyl sulfide,a mustard analog

2‐Choloroethyl Ethyl Sulfide (CEES) exposure causes inflammatory lung diseases, including acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. This may be associated with oxidative stress, which has been implicated in the desensitization of beta‐adrenergic receptors (β‐ARs). The objective of this study was to investigate whether lung injury induced by intratracheal CEES exposure (2 mg/kg body weight) causes desensitization of β‐ARs. The animals were sacrificed after 7 days and lungs were removed. Lung injury was established by measuring the leakage of iodinated‐bovine serum albumin ([¹²⁵I]‐BSA) into lung tissue. Receptor‐binding characteristics were determined by measuring the binding of [³H] dihydroalprenolol ([³H] DHA) (0.5–24 nM) to membrane fraction in the presence and absence of DLDL ‐propranolol (10 μ M). Both high‐ and low‐affinity β‐ARs were identified in the lung. Binding capacity was significantly higher in low‐affinity site in both control and experimental groups. Although CEES exposure did not change K_D and B_max at the high‐affinity site, it significantly decreased both K_D and B_max at low affinity sites. A 20% decrease in β₂‐AR mRNA level and a 60% decrease in membrane protein levels were observed in the experimental group. Furthermore, there was significantly less stimulation of adenylate cyclase activity by both cholera toxin and isoproterenol in the experimental group in comparison to the control group. Treatment of lungs with 3‐isobutyl‐1‐methylxanthine (IBMX), an inhibitor of phosphodiesterase (PDE) could not abolish the difference between the control group and the experimental group on the stimulation of the adenylate cyclase activity. Thus, our study indicates that CEES‐induced lung injury is associated with desensitization of β₂‐AR. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:59–70, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20265     

Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (K_D > 10⁻⁸ M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K_D = 1.1 × 10⁻¹⁰ M and K_D = 3.34 × 10⁻¹⁰ M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Förster resonance energy transfer measurements of cofactor‐dependent effects on protein arginine N‐methyltransferase homodimerization

Protein arginine N‐methyltransferase (PRMT) dimerization is required for methyl group transfer from the cofactor S‐adenosyl‐L ‐methionine (AdoMet) to arginine residues in protein substrates, forming S‐adenosyl‐L ‐homocysteine (AdoHcy) and methylarginine residues. In this study, we use Förster resonance energy transfer (FRET) to determine dissociation constant (K_D) values for dimerization of PRMT1 and PRMT6. By attaching monomeric Cerulean and Citrine fluorescent proteins to their N‐termini, fluorescent PRMTs are formed that exhibit similar enzyme kinetics to unconjugated PRMTs. These fluorescent proteins are used in FRET‐based binding studies in a multi‐well format. In the presence of AdoMet, fluorescent PRMT1 and PRMT6 exhibit 4‐ and 6‐fold lower dimerization K_D values, respectively, than in the presence of AdoHcy, suggesting that AdoMet promotes PRMT homodimerization in contrast to AdoHcy. We also find that the dimerization K_D values for PRMT1 in the presence of AdoMet or AdoHcy are, respectively, 6‐ and 10‐fold lower than the corresponding values for PRMT6. Considering that the affinity of PRMT6 for AdoHcy is 10‐fold higher than for AdoMet, PRMT6 function may be subject to cofactor‐dependent regulation in cells where the methylation potential (i.e., ratio of AdoMet to AdoHcy) is low. Since PRMT1 affinity for AdoMet and AdoHcy is similar, however, a low methylation potential may not affect PRMT1 function.     

Brain region‐specific changes in N‐acylethanolamine contents with time of day

The N‐acylethanolamines (NAEs) exert important behavioral, physiological, and immunological effects through actions at cannabinoid and other receptors. We measured concentrations of three NAEs, the K_m and V_max for fatty acid amide hydrolysis (FAAH), FAAH protein and FAAH mRNA in prefrontal cortex, hippocampus, hypothalamus, amygdala, striatum, and cerebellum at 4 h intervals, starting at 03:00. Significant differences in N‐arachidonylethanolamine contents among the times examined occur in the prefrontal cortex (PFC), hippocampus, hypothalamus, and striatum. N‐Oleoylethanolamine concentrations exhibit large fluctuations over the day in the cerebellum, including a threefold decrease between 19:00 and 23:00. N‐Palmitoylethanolamine and N‐oleoylethanolamine were significantly, positively correlated in all regions examined except the hypothalamus. FAAH K_m values are significantly affected by time of day in PFC, hippocampus and amygdala and FAAH V_max values are significantly affected in PFC, hippocampus and cerebellum. However, correlational data indicate that FAAH does not play a primary role in the circadian regulation of the NAE concentrations. FAAH protein expression is not significantly different among the harvest times in any brain region examined. Concentrations of 2‐arachidonoylglycerol are significantly affected by time of harvest in the striatum and cerebellum, but not in other brain regions. Together, these data indicate that the NAEs exhibit diverse patterns of change with time of day that are likely the result of alterations in biosynthesis, and support the hypothesis that N‐arachidonylethanolamine is a tonic activator of cannabinoid receptor signaling.

     

Principal component analysis of chemical shift perturbation data of a multiple‐ligand‐binding system for elucidation of respective binding mechanism

Chemical shift perturbations (CSPs) in NMR spectra provide useful information about the interaction of a protein with its ligands. However, in a multiple‐ligand‐binding system, determining quantitative parameters such as a dissociation constant (K_d) is difficult. Here, we used a method we named CS‐PCA, a principal component analysis (PCA) of chemical shift (CS) data, to analyze the interaction between bovine β‐lactoglobulin (βLG) and 1‐anilinonaphthalene‐8‐sulfonate (ANS), which is a multiple‐ligand‐binding system. The CSP on the binding of ANS involved contributions from two distinct binding sites. PCA of the titration data successfully separated the CSP pattern into contributions from each site. Docking simulations based on the separated CSP patterns provided the structures of βLG–ANS complexes for each binding site. In addition, we determined the K_d values as 3.42 × 10⁻⁴M² and 2.51 × 10⁻³M for Sites 1 and 2, respectively. In contrast, it was difficult to obtain reliable K_d values for respective sites from the isothermal titration calorimetry experiments. Two ANS molecules were found to bind at Site 1 simultaneously, suggesting that the binding occurs cooperatively with a partial unfolding of the βLG structure. On the other hand, the binding of ANS to Site 2 was a simple attachment without a significant conformational change. From the present results, CS‐PCA was confirmed to provide not only the positions and the K_d values of binding sites but also information about the binding mechanism. Thus, it is anticipated to be a general method to investigate protein–ligand interactions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Atomistic mechanisms of huntingtin N‐terminal fragment insertion on a phospholipid bilayer revealed by molecular dynamics simulations

The huntingtin protein is characterized by a segment of consecutive glutamines (Q_N) that is responsible for its fibrillation. As with other amyloid proteins, misfolding of huntingtin is related to Huntington's disease through pathways that can involve interactions with phospholipid membranes. Experimental results suggest that the N‐terminal 17‐amino‐acid sequence (htt^NT) positioned just before the Q_N region is important for the binding of huntingtin to membranes. Through all‐atom explicit solvent molecular dynamics simulations, we unveil the structure and dynamics of the htt^NTQ_N fragment on a phospholipid membrane at the atomic level. We observe that the insertion dynamics of this peptide can be described by four main steps—approach, reorganization, anchoring, and insertion—that are very diverse at the atomic level. On the membrane, the htt^NT peptide forms a stable α‐helix essentially parallel to the membrane with its nonpolar side‐chains—mainly Leu‐4, Leu‐7, Phe‐11 and Leu‐14—positioned in the hydrophobic core of the membrane. Salt‐bridges involving Glu‐5, Glu‐12, Lys‐6, and Lys‐15, as well as hydrogen bonds involving Thr‐3 and Ser‐13 with the phospholipids also stabilize the structure and orientation of the htt^NT peptide. These observations do not significantly change upon adding the Q_N region whose role is rather to provide, through its hydrogen bonds with the phospholipids' head group, a stable scaffold facilitating the partitioning of the htt^NT region in the membrane. Moreover, by staying accessible to the solvent, the amyloidogenic Q_N region could also play a key role for the oligomerization of htt^NTQ_N on phospholipid membranes. Proteins 2014; 82:1409–1427. © 2014 Wiley Periodicals, Inc.     

Three‐dimensional structure,binding, and spectroscopic characteristics of the monoclonal antibody 43.1 directed to the carboxyphenyl moiety of fluorescein

Unlike other known anti‐fluorescein antibodies, the monoclonal antibody 43.1 is directed toward the fluorescein's carboxyl phenyl moiety. It demonstrates a very high affinity (K_D ～ 70 pM) and a fast association rate (k_on ～ 2 × 10⁷ M^?1 s^?1). The three‐dimensional structure of the Fab 43.1—fluorescein complex was resolved at 2.4 Å resolution. The antibody binding site is exclusively assembled by the CDR loops. It is comprised of a 14 Å groove‐shaped entrance leading to a 9 Å by 7 Å binding pocket. The highly polar binding pocket complementary encloses the fluorescein's carboxyphenyl moiety and tightly fixes it by multiple hydrogen bonds. The fluorescein's xanthene ring is embedded in the more hydrophobic groove and stacked between the side chains of Tyr37_L and of Arg99_H providing conditions for an excited state electron transfer process. In comparison to fluorescein, the absorption spectrum of the complex in the visible region is shifted to the “red” by 23 nm. The complex demonstrates a very weak fluorescence (Φ_c = 0.0018) with two short lifetime components: 0.03 ns (47%) and 0.8 ns (24%), which reflects a 99.8% fluorescein emission quenching effect upon complex formation. The antibody 43.1 binds fluorescein with remarkable affinity, fast association rate, and strongly quenches its emission. Therefore, it may present a practical interest in applications such as molecular sensors and switches. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 234–243, 2016.     

Candidacidal mechanism of a Leu/Lys‐rich α‐helical amphipathic model antimicrobial peptide and its diastereomer composed of D,L‐amino acids

We investigated the mechanism of candidacidal action of a Lys/Leu‐rich α‐helical model antimicrobial peptide (K₉L₈W) and its diastereomeric peptide (D₉‐K₉L₈W) composed of D ,L ‐amino acids. K₉L₈W killed completely Candida albicans within 30 min, but D₉‐K₉L₈W killed only 72% of C. albicans even after 100 min. Tryptophan fluorescence spectroscopy indicated that the fungal cell selectivity of D₉‐K₉L₈W is closely correlated with a selective interaction with the negatively charged PC/PE/PI/ergosterol (5:2.5:2.5:1, w/w/w/w) phospholipids, which mimic the outer leaflet of the plasma membrane of C. albicans. K₉L₈W was able to induce almost 100% calcein leakage from PC/PE/PI/ergosterol (5:2.5:2.5:1, w/w/w/w) liposomes at a peptide:lipid molar ratio of 1:16, whereas D₉‐K₉L₈W caused only 25% dye leakage even at a peptide:lipid molar ratio of 1:2. Confocal laser‐scanning microscopy revealed that FITC‐labeled D₉‐K₉L₈W penetrated the cell wall and cell membrane and accumulated inside the cells, whereas FITC‐labeled K₉L₈W did not penetrate but associated with the membranes. Collectively, our results demonstrated that the candidacidal activity of K₉L₈ W and D₉‐K₉L₈W may be due to the transmembrane pore/channel formation or perturbation of the fungal cytoplasmic membranes and the inhibition of intracellular functions, respectively. Finally, D₉‐K₉L₈W with potent anti‐Candida activity but no hemolytic activity may be potentially a useful lead compound for the development of novel antifungal agents. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Oligohis‐tags: mechanisms of binding to Ni2+‐NTA surfaces

Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni²⁺ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C‐ or N‐terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His‐tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His‐tags to Ni²⁺‐NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid‐phase peptide synthesis (SPPS). Binding to Ni²⁺‐NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (K_D) of 14 ± 1 nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative kinetics of fatty acid-amino acid conjugate elicitor biosynthesis by midgut tissue microsomes of Lepidopterous caterpillar larvae

N‐Linolenoyl‐L ‐glutamine is one of several structurally similar fatty acid–amino acid conjugate (FAC) elicitors found in the oral secretions of Lepidopterous caterpillars and its biosynthesis is catalyzed by membrane‐associated alimentary tissue enzyme(s). FAC elicitors comprise 17‐hydroxylated or non‐hydroxylated linolenic acid coupled with L ‐glutamine or L ‐glutamate by an amide bond. We demonstrate in vitro biosynthesis of N‐linolenoyl‐L ‐glutamine by Manduca sexta, Heliothis virescens, and Helicoverpa zea tissue microsomes. Comparison of N‐linolenoyl‐L ‐glutamine biosynthesis kinetics for these species suggests that concurrent biosynthesis and hydrolysis contribute to proportions of FAC elicitors found in their oral secretions. The apparent K_m values for coupling of sodium linolenate were 8.75±0.79, 14.3±3.7 and 20.7±3.4 mM and V_max values were 2.92±0.14, 6.81±1.2 and 4.95±0.55 nmol/min/mg protein for H. zea, H. virescens and M. sexta, respectively. The K_m values for coupling of L ‐glutamine were 10.5±0.26, 22.3±2.0 and 18.9±2.4 mM and V_max values were 1.78±0.21, 3.71±0.50 and 2.49±0.41 nmol/min/mg of protein for H. zea, H. virescens and M. sexta, respectively. © 2010 Wiley Periodicals, Inc.     

Detoxification of insecticides,allechemicals and heavy metals by glutathione S‐transferase SlGSTE1 in the gut of Spodoptera litura

Insect glutathione S‐transferases (GSTs) play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST SlGSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. SlGSTE1 was significantly up‐regulated by chlorpyrifos and xanthotoxin in the midgut of S. litura. The recombinant SlGSTE1 had V_max (reaction rate of the enzyme saturated with the substrate) and K_m (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) values of 27.95 ± 0.88 μmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respectively, and V_max and K_m values of 22.96 ± 0.78 μmol/min/mg and 0.83 ± 0.106 mmol/L for 1‐chloro‐2,4‐dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant SlGSTE1 possessed high binding activities to the insecticides chlorpyrifos, deltamethrin, malathion, phoxim and dichloro‐diphenyl‐trichloroethane (DDT). SlGSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that SlGSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. SlGSTE1 also showed high peroxidase activity. All the results together indicate that SlGSTE1 may play an important role in the gut of S. litura to protect the insect from the toxic effects of these compounds and heavy metals.