Schiff Base Linkage Research Articles

Glutaraldehyde-Activated Chitosan Matrix for Immobilization of a Novel Cysteine Protease, Procerain B

Proteases have several applications in the food industry. We report the immobilization of procerain B, a novel cysteine protease, on glutaraldehyde-activated chitosan beads through covalent attachment. Glutaraldehyde not only serves as a cross-linking agent but also links the procerain B on the surface of bead through primary amine group (either lysine side chain or N-terminal) by Schiff base linkage. Immobilized procerain B was characterized for optimum functional range and stability with respect to pH and temperature. The chitosan-immobilized procerain B has broad pH and thermal optima. The effects of substrate concentration and reusability of immobilized beads were also studied. It showed nearly 50% activity until the 10th use.

Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry

We report the application of pulsed oxidative labeling for deciphering the folding mechanism of a membrane protein. SDS-denatured bacteriorhodopsin (BR) was refolded by mixing with bicelles in the presence of free retinal. At various time points (20 ms to 1 day), the protein was exposed to a microsecond ·OH pulse that induces oxidative modifications at solvent-accessible methionine side chains. The extent of labeling was determined by mass spectrometry. These measurements were complemented by stopped-flow spectroscopy. Major time-dependent changes in solvent accessibility were detected for M20 (helix A) and M118 (helix D). Our kinetic data indicate a sequential folding mechanism, consistent with models previously suggested by others on the basis of optical data. Yet, ·OH labeling provides additional structural insights. An initial folding intermediate I 1 gets populated within 20 ms, concomitantly with formation of helix A. Subsequent structural consolidation leads to a transient species I 2. Noncovalent retinal binding to I 2 induces folding of helix D, thereby generating an intermediate I R⁎. In the absence of retinal, the latter transition does not take place. Hence, formation of helix D depends on retinal binding, whereas this is not the case for helix A. As the cofactor settles deeper into its binding pocket, a final transient species I R⁎⁎ is generated. This intermediate converts into native BR within minutes by formation of the retinal–K216 Schiff base linkage. The combination of pulsed covalent labeling and optical spectroscopy employed here should also be suitable for exploring the folding mechanisms of other membrane proteins.

Crystal structure of metarhodopsin II

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0 Å and 2.85 Å crystal structures, respectively, of Meta II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta II structures, the electron density from the retinal ligand seamlessly continues into the Lys 296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta II. The structures can now serve as models for the large GPCR family.

Thermal Stability of Rhodopsin and Implications for Retinitis Pigmentosa

Over 100 mutations in the dim-light photoreceptor rhodopsin are associated with retinitis pigmentosa (RP), a visual disorder characterized by progressive symptoms of night blindness, tunnel vision, and sometimes blindness. We studied the thermal stability of two mutations, S186W and D190N. We hypothesize that these mutations perturb an electrostatic interaction and hydrogen bonds in the active site and destabilize rhodopsin, which undermines the sensitivity to light and causes RP. We compared the rates of thermal decay, thermal isomerization, and Schiff base hydrolysis of WT, S186W, and D190N rhodopsin. Using UV-visible spectroscopy, we observed that the D190N mutant and WT rhodopsin do not decay over 24 hours at 37°C, whereas the S186W mutant decays with a half-life of 36 ± 4 min. We also measured the half-lives at 55°C, which are 70 ± 2 min for WT, 2.4 ± 0.2 for D190N, and 0.43 ± 0.03 min for S186W. Using HPLC and the acid denaturation assay, we measured the rates of thermal isomerization of 11-cis retinal and hydrolysis of the Schiff base linkage between retinal and opsin. We found that the mutations also increase these rates by 1-2 orders of magnitude. Because thermal isomerization of rhodopsin generates the same physiological response as photoisomerization, we suggest that the higher thermal isomerization rate in the mutants increases the level of dark noise, which desensitizes rhodopsin and causes the early symptom of night blindness. Because the drastic destabilizing effect of the mutations is likely correlated with the progressive deformation of the outer segment and subsequent loss of rod cells in RP, we propose that a future systematic study of the thermal stability of the RP-causing mutations can potentially provide more accurate predictions of the pace of vision loss in patients and guide strategies for treatment.

Enhanced molecular chaperone activity of the small heat‐shock protein αB‐crystallin following covalent immobilization onto a solid‐phase support

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.

Degradable and biocompatible aldehyde-functionalized glycopolymer conjugated with doxorubicin via acid-labile Schiff base linkage for pH-triggered drug release

Well-defined aldehyde-functionalized glycopolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) copolymerization of 1,2:3,4-di-O-isopropylidene-6-O-(2′-formyl-4′-vinylphenyl)-D-galactopyranose (IVDG) and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) using dicumyl peroxide as the initiator and 1-phenylethyl phenyldithioacetate as the RAFT agent at 130 °C in anisole. The resulting copolymers were found to be hydrolytically degradable due to their main-chain polyester structures. Removal of the protective isopropylidene groups from the sugar residues resulted in a novel amphiphilic copolymer with low cytotoxicity as confirmed by MTT assay against L929 cells. The deprotected copolymer could conjugate anticancer drug DOXvia an acid-labile Schiff base linkage to form DOX-loaded micelles in a high drug loading level (∼14 wt%) with pendent galactose moieties covering the surface. The size of the DOX-conjugated polymeric micelle was determined to be about 125 nm by dynamic light scattering. The in vitro release studies demonstrated that the release of DOX from the micelles manifested a strong dependence on the environment pH due to the acid-cleavable Schiff base linkage between the DOX and micelles. The DOX release was significantly faster at pH 5.0 compared to pH 7.4.

Cholesterol-based unsymmetrical Schiff's base dimer terminated with 4-alkoxy-5-phenylthiophene unit: synthesis and characterisation

A series of unsymmetrical cholesterol based dimers have been synthesised and characterised. All the dimers possess a cholesterol unit connected with a 4-alkoxy-5-phenylthiophene unit through Schiff's base linkage. The length of the terminal alkyl and the alkyl spacer has been varied. All the dimers exhibit mesomorphism. Four dimers possess only the cholesteric (chiral nematic, N*) phase. One of the dimers showed smectic A (SmA) and N* phases whereas the other dimer displayed SmA, smectic chiral C (SmC*), N* and twist grain boundary (TGB) phases.

Photosensitivities of Rhodopsin Mutants with a Displaced Counterion

Visual pigments consist of a protein moiety opsin and an 11-cis-retinal chromophore that is covalently bound to the opsin via a Schiff base linkage. They have a high photosensitivity, which can be attributed to the high probability of photon absorption and the high photoisomerization quantum yield of the retinal chromophore. Both of these parameters are regulated by the opsin, though the precise mechanism is unknown. We previously found that counterion residue E113, which stabilizes the proton on the Schiff base, is involved in the efficient photoisomerization in vertebrate visual pigments. To test the positional effect of the counterion on the photon absorption and the photoisomerization, we measured the photosensitivities of a set of mutants of bovine rhodopsin in which the counterion was displaced to position 90, 94, 117, or 292. The molar extinction coefficient was reduced in many of the mutants, leading to reductions in the photosensitivity for monochromatic lights. However, the oscillator strength, the probability of photon absorption integrated over the entire wavenumber range of the absorption band, was relatively similar among the mutants and the wild type. In addition, the quantum yields of the mutants were not markedly different from that of the wild type. These results indicate that the counterion does not need to be located at position 113 for a high photosensitivity for natural light. Interestingly, all of the mutants exhibited greatly increased hydroxylamine sensitivity. This result suggests that the counterion in vertebrate visual pigments is optimally located for the stability of the Schiff base linkage.

Identification of the Structural Determinants for the Stability of Substrate and Aminoacrylate External Schiff Bases in O-Acetylserine Sulfhydrylase-A

O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the beta-acetoxy group of O-acetyl-L-serine (OAS) by a thiol to give L-cysteine. Previous studies of the K41A mutant enzyme showed L-methionine bound in an external Schiff base (ESB) linkage to PLP as the enzyme was isolated. The mutant enzyme exists in a closed form, optimizing the orientation of the cofactor PLP and properly positioning active site functional groups for reaction. The trigger for closing the active site upon formation of the ESB is thought to be interaction of the substrate alpha-carboxylate with the substrate-binding loop comprised of T68, S69, G70, and N71, and Q142, which is positioned above the cofactor as one looks into the active site. To probe the contribution of these residues to the active site closing and orientation of PLP in the ESB, T68, S69, N71, and Q142 were changed to alanine. Absorbance, fluorescence, near UV-visible CD, and (31)P NMR spectral studies and pre-steady state kinetic studies were used to characterize the mutant enzymes. All of the mutations affect closure of the active site, but to differing extents. In addition, the site appears to be more hydrophilic given that the ESBs do not exhibit a significant amount of the enolimine tautomer. No buildup of the alpha-aminoacrylate intermediate (AA) is observed for the T68A and Q142A mutant enzymes. However, pyruvate is produced at a rate much greater than that of the wild-type enzyme. Data suggest that T68 and Q142 play a role in stabilizing the AA. Both residues donate a hydrogen bond to one of the carboxylate oxygens of the methionine ESB and may also be responsible for the proper orientation of the ESB to generate the AA. The S69A and N71A mutants exhibit formation of the AA, but the rate constant for its formation from the ESB is decreased by 1 order of magnitude compared to that of the wild type. S69 donates a hydrogen bond to the substrate carboxylate in the ESB, while N71 donates hydrogen bonds to O3' of the cofactor and the carboxylate of the ESB; these side chains may also affect the orientation of the ESB. Data suggest that interaction of intermediates with the substrate-binding loop and Q142 gives a properly aligned Michaelis complex and facilitates the beta-elimination reaction.

FTIR Study of the Photoreaction of Bovine Rhodopsin in the Presence of Hydroxylamine

In bovine rhodopsin, 11-cis-retinal forms a Schiff base linkage with Lys296. The Schiff base is not reactive to hydroxylamine in the dark, which is consistent with the well-protected retinal binding site. In contrast, under illumination it easily forms all-trans retinal oxime, resulting in the loss of color. This suggests that activation of rhodopsin creates a specific reaction channel for hydroxylamine or loosens the chromophore binding pocket. In the present study, to extract structural information on the Schiff base vicinity and to understand the changes upon activation of rhodopsin, we compared light-induced FTIR difference spectra of bovine rhodopsin in the presence and absence of hydroxylamine under physiological pH (approximately 7). Although the previous FTIR study did not observe the complex formation between rhodopsin and G-protein transducin in hydrated films, the present study clearly shows that hydrated films can be used for studies of the interaction between rhodopsin and hydroxylamine. Hydroxylamine does not react with the Schiff base of Meta-I intermediate trapped at 240 K, possibly because of decreased conformational motions under the frozen environment, while FTIR spectroscopy showed that hydroxylamine affects the hydrogen bonds of the Schiff base and water molecules in Meta-I. In contrast, formation of the retinal oxime was clearly observed at 280 K, the characteristic temperature of Meta-II accumulation in the absence of hydroxylamine, and time-dependent formation of retinal oxime was observed from Meta-II at 265 K as well. The obtained difference FTIR spectra of retinal oxime and opsin are different from that of Meta-II. It is likely that the antiparallel beta-sheet constituting a part of the retinal binding pocket at the extracellular surface is structurally disrupted in the presence of hydroxylamine, which allows the hydrolysis of the Schiff base into retinal oxime.

Synthesis of new schiff base ester liquid crystals with a benzothiazole core

A series of new calamitic liquid crystals, 6-methoxy-2-(4-alkanoyloxybenzylidenamino)benzothiazoles, comprising a benzothiazole core, terminal methoxy group and a Schiff base linkage were synthesised and characterised. This series comprises 12 members wherein members differ by the length of the alkanoyloxy chain (C n-1H2n-1COO-, where n = 2–8, 10, 12, 14, 16, 18). Their mesomorphic properties were studied by using differential scanning calorimetry, optical polarising microscopy and powder X-ray diffraction techniques. The short chain derivatives (n = 2 and 3) were non-mesogenic compounds, while an enantiotropic nematic phase was present throughout the remaining members of the series. The smectic C phase emerged from the decanoyloxy derivative onwards.

Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

The hydrophobic nature of membrane proteins poses considerable challenges for the application of traditional analytical techniques. Hydrogen/deuterium exchange (HDX) with electrospray mass spectrometry (ESI-MS) detection is a potentially very powerful approach for investigating the structure and dynamics of membrane proteins, but thus far this technique has been applied mostly to soluble species. By using ESI-MS in conjunction with low temperature size exclusion chromatography (SEC) the current study explores the global HDX behavior of the integral membrane protein bacteriorhodopsin (BR) under various conditions. The experiments are complemented by UV–vis absorption measurements that report on the environment of the protein's retinal chromophore and fluorescence spectroscopy which probes the extent of resonance energy transfer between Trp residues and retinal. In agreement with previous reports, our HDX data imply that amide hydrogens in the transmembrane helices of native BR are highly protected, whereas most sites in solvent-exposed loops are readily exchangeable. Low temperature SEC/ESI-MS allows the labile Schiff-base linkage between protein and retinal to be completely preserved, such that the HDX properties of co-existing protein populations can be monitored individually. Solubilization of BR in sodium dodecyl sulfate (SDS) induces major structural changes and hydrolytic loss of retinal. The SDS-denatured protein appears to retain a significant degree of stable helical structure (around 25%, compared to the native state value of 74%). Bicelle-mediated refolding of SDS-denatured bacterioopsin in the presence of free retinal regenerates a native-like BR conformation with a yield close to 0.9. The HDX/SEC/ESI-MS approach developed in this work should be applicable to other membrane protein systems as well.

Chiral Discrimination among Aminotransferases: Inactivation by 4-Amino-4,5-dihydrothiophenecarboxylic Acid

Mechanism-based inhibitors such as cycloserine and gabaculine can inactivate aminotransferases via reactions of the compounds with the pyridoxal phosphate cofactor forming an irreversible adduct. The reaction is chirally specific in that any one enzyme usually only recognizes one enantiomer of the inactivator. For instance, l-aspartate aminotransferase (l-AspAT) is inactivated by 4-amino-4,5-dihydro-2-thiophenecarboxylic acid (ADTA), however, only by the S-isomer. We have now shown that d-amino acid aminotransferase (d-a-AT) is irreversibly inactivated by the R-isomer of the same compound. The X-ray crystal structure (PDB code: 3LQS ) of the inactivated enzyme shows that in the product the enzyme no longer makes a Schiff base linkage to the pyridoxal 5'-phosphate (PLP) cofactor, and instead the compound has formed a derivative of the cofactor. The adduct is similar to that formed between d-cycloserine and d-a-AT or alanine racemase (Ala-Rac) in that the thiophene ring of R-ADTA is intact and seems to be aromatic. The plane of the ring is rotated by nearly 90 degrees with respect to the plane of the pyridine ring of the cofactor, in comparison with the enzyme inactivated by cycloserine. Based on the structure of the product, the mechanism of inactivation most probably involves a transamination followed by aromatization to form an aromatic thiophene ring.

Gelatin hydrogels with PAMAM nanostructured surface and high density surface-localized amino groups

Amino-terminated PAMAM dendrimers with 16 and respectively 64 amino groups have been chemically immobilized onto the surface of glutaraldehyde (abbreviated GA) crosslinked thin gelatin scaffolds based on Schiff-base linkage formation between the amines and free aldehyde from partially unreacted GA. Crosslinking and dendrimers surface modification of hydrogels have been chemically and physically investigated using FT-IR spectroscopy and microscopy, UV–VIS, LM, AFM, swelling tests and a colorimetric technique. Two types of gelatin-based hydrogels with homogeneous nanorough surfaces and presenting an increased number of amino groups available for further chemical reactions have been obtained. The synthesis strategy presented in this work seems to open a new route for nanorough crosslinked gelatin scaffolds that could be further used for biomedical and especially for hard tissue engineering application.

Novel Enzymatic Function of DNA Polymerase ν in Translesion DNA Synthesis Past Major Groove DNA−Peptide and DNA−DNA Cross-Links

DNA polymerase ν (POLN or pol ν) is a newly discovered A family polymerase that generates a high error rate when incorporating nucleotides opposite dG; its translesion DNA synthesis (TLS) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions. In the current investigation, we describe a novel TLS substrate specificity of pol ν, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol ν catalyzed efficient and high fidelity TLS past peptides linked to N6-dA via a reduced Schiff base linkage with a γ-hydroxypropano-dA. Additionally, pol ν could bypass DNA interstrand cross-links with linkage between N6-dAs in complementary DNA strands. However, the chemically identical DNA−peptide and DNA interstrand cross-links completely blocked pol ν when they were located in the minor groove via a N2-dG linkage. Furthermore, we showed that pol ν incorporated a nucleotide opposite the 1,N6-etheno-dA (εdA) in an error-free manner and (+)-trans-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-dA [(+)-BPDE-dA] in an error-prone manner, albeit with a greatly reduced capability. Collectively, these data suggest that although pol ν bypass capacity cannot be generalized to all major groove DNA adducts, this polymerase could be involved in TLS when genomic replication is blocked by extremely large major groove DNA lesions. In view of the recent observation that pol ν may have a role in cellular tolerance to DNA cross-linking agents, our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some DNA−protein and DNA−DNA cross-links.

Direct Observation of the pH-Dependent Equilibrium between Metarhodopsins I and II and the pH-Independent Interaction of Metarhodopsin II with Transducin C-Terminal Peptide

Bovine rhodopsin contains 11-cis-retinal as a light-absorbing chromophore that binds to a lysine residue of the apoprotein opsin via a protonated Schiff base linkage. Light isomerizes 11-cis-retinal into the all-trans form, which eventually leads to the formation of an enzymatically active state, metarhodopsin II (MII). It is widely believed that MII forms a pH-dependent equilibrium with metarhodopsin I (MI), but direct evidence for this equilibrium has not been reported. Here, we confirmed this equilibrium by direct observation of the mutual conversions of MI and MII upon changing the pH of the MI/MII mixture. We also observed a reversible binding of the synthetic peptide constituting the C-terminal 11 amino acids of the transducin alpha-subunit to MII, which resulted in change of the amounts of MI and MII in the equilibrium. Interestingly, addition of the peptide did not induce a simple pK(a) shift but rather induced an increase of the MII fraction at high pH. These results indicate that in addition to the MII that is formed from MI in a pH-dependent manner there also exists another MII, which is in equilibrium with MI in a pH-independent manner and can bind to the peptide. Therefore, there is no need for proton uptake by the protein moiety of opsin for the binding to the peptide.

NinaB Is Essential for Drosophila Vision but Induces Retinal Degeneration in Opsin-deficient Photoreceptors

In animals, visual pigments are essential for photoreceptor function and survival. These G-protein-coupled receptors consist of a protein moiety (opsin) and a covalently bound 11-cis-retinylidene chromophore. The chromophore is derived from dietary carotenoids by oxidative cleavage and trans-to-cis isomerization of double bonds. In vertebrates, the necessary chemical transformations are catalyzed by two distinct but structurally related enzymes, the carotenoid oxygenase beta-carotenoid-15,15'-monooxygenase and the retinoid isomerase RPE65 (retinal pigment epithelium protein of 65 kDa). Recently, we provided biochemical evidence that these reactions in insects are catalyzed by a single enzyme family member named NinaB. Here we show that in the fly pathway, carotenoids are mandatory precursors of the chromophore. After chromophore formation, the retinoid-binding protein Pinta acts downstream of NinaB and is required to supply photoreceptors with chromophore. Like ninaE encoding the opsin, ninaB expression is eye-dependent and is activated as a downstream target of the eyeless/pax6 and sine oculis master control genes for eye development. The requirement for coordinated synthesis of chromophore and opsin is evidenced by analysis of ninaE mutants. Retinal degeneration in opsin-deficient photoreceptors is caused by the chromophore and can be prevented by restricting its supply as seen in an opsin and chromophore-deficient double mutant. Thus, our study identifies NinaB as a key component for visual pigment production and provides evidence that chromophore in opsin-deficient photoreceptors can elicit retinal degeneration.

Rpe65 Isomerase Associates with Membranes through an Electrostatic Interaction with Acidic Phospholipid Headgroups

Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.

K30, H150, and H168 Are Essential Residues for Coordinating Pyridoxal 5′-Phosphate of O-Acetylserine Sulfhydrylase from Acidithiobacillus ferrooxidans

O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV–vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5′-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization.

Cyclic dithia/diaza with dual Schiff base linkage functionalized polymers for heavy metal adsorption

AbstractThree new types of functionalized polystyrene‐divinylbenzene resins containing two sulfur and two nitrogen atoms and four nitrogen atoms in cyclic form were prepared by using two lab‐synthesized ligands; 2‐[3‐(2‐amino ethylsulfanyl)‐propylsulfanyl]‐ethylamine (AEPE) and 2‐[2‐(2‐amino‐ethylsulfanyl)‐ethylsulfanyl]‐ethylamine (AEEE), and a commercial one; triethylenetetramine (TETA). Chloromethylated polystyrene‐divinylbenzene copolymer (Cl‐PS‐DVB) was initially oxidized to aldehydic polystyrene‐divinylbenzene (CHO‐PS‐DVB) with approximately 57% yield. CHO‐PS‐DVB was subsequently reacted with the ligands through dual Schiff base linkage. The products were named AEPE‐PS‐DVB, AEEE‐PS‐DVB and TETA‐PS‐DVB, respectively. The optimum coupling reaction times and temperatures were 6 h at 50°C for AEPE‐PS‐DVB, AEEE‐PS‐DVB and 6 h at room temperature for TETA‐PS‐DVB. The appropriate amount of the three ligands was 2.65 mmol/g CHO‐PS‐DVB corresponding to the mole ratio between the chelating ligand and aldehyde group of 1 : 2. All derivative resins were characterized by elemental analysis, thermogravimetry, FT‐IR and FT‐Raman spectroscopy and Ninhydrin test. The adsorption properties of the synthesized resins towards Pb(II), Cu(II), Cd(II), Zn(II), Ni(II), Co(II) and Cr(III) ions in aqueous solution were preliminarily investigated by batch method. AEPE‐PS‐DVB and AEEE‐PS‐DVB showed the same sorption behavior. Their selectivity order was Pb(II) > Zn(II) > Cu(II) > Cd(II) and no significant adsorption for Co(II), Ni(II), Cr(III) was observed. In addition, TETA‐PS‐DVB showed a selectivity toward only Pb(II). © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010