Affinity dye–ligand poly(hydroxyethyl methacrylate)/chitosan composite membrane for adsorption lysozyme and kinetic properties

A composite membrane from 2-hydroxyethyl methacrylate (HEMA) and poly(hydroxyethyl methacrylate)/chitosan (pHEMA/chitosan) was synthesized via UV initiated photo-polymerization in the presence of an initiator α,α′-azoisobutyronitrile (AIBN). Procion Brown MX 5BR was then covalently immobilized onto composite membrane as a dye–ligand. The binding characteristics of a model protein (i.e. lysozyme) to the dye–ligand immobilized affinity membrane have been investigated from aqueous solution using the plain composite membrane as a control system. The experimental data was analyzed using two adsorption kinetic models, the pseudo-first-order and the pseudo-second-order, to determine the best-fit equation for the adsorption of lysozyme onto affinity composite membrane. The second-order equation for the adsorption of lysozyme on the dye–ligand membrane systems is the most appropriate equation to predict the adsorption capacity for the affinity membrane. The reversible lysozyme adsorption on the affinity membrane obeyed the Freundlich isotherm. The lysozyme adsorption capacity of the plain membrane and the dye–ligand affinity membrane were 8.3 and 121.5 mg ml⁻¹, respectively.     

Probing the Interaction of Human Serum Albumin With Bilirubin in the Presence of Aspirin by Multi-Spectroscopic,Molecular Modeling and Zeta Potential Techniques: Insight on Binary and Ternary Systems

Abstract

Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (K_a) and number of binding site (n) obtained were 1.6 × 10⁶ M^?1 and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 10⁶ M^?1 and 0.84, respectively, in the presence of ASA (ternary complex) at δ_ex = 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (C_CIAC) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.     

Hydroxylamine as a thermal destabiliser of bacteriorhodopsin

The light-catalysed reaction of hydroxylamine (HA) with retinal is one of the basic features of bacteriorhodopsin (BR). Surprisingly, according to recent results, neither the photocycle and proton pumping of BR, nor the trans–cis isomerisation of retinal is prerequisite for photobleaching of BR in the presence of HA. How, then, is the accessibility of retinal to HA enhanced on illumination? We studied whether local thermal denaturation of BR, proposed recently, could provide an explanation for HA-promoted bleaching. According to our results, HA does not alter the absorption spectrum and the photocycle kinetics of BR substantially at room temperature, even at molar concentrations, but grossly affects the temperature of thermal denaturation. At pH 7, the presence of 0.5 M HA reduces the denaturation temperature from 100°C to as low as 72°C. The decrease is proportional to the logarithm of the HA concentration over more than three orders of magnitude, and even 0.5 mM HA has a significant effect. In addition, photobleaching becomes considerably faster with increasing temperature in the presence of HA, it takes a few seconds at 50–60°C. Our results suggest that photobleaching of BR in the presence of HA can be explained by overall destabilisation of the structure of the protein and local thermal denaturation that has already accounted for the photobleaching of the HA-free BR at elevated temperatures. These results further support the importance of thermooptic effects in protein photoreactions and identify HA as a thermal destabiliser of BR.     

A Comprehensive Insight into Binding of Hippuric Acid to Human Serum Albumin: A Study to Uncover Its Impaired Elimination through Hemodialysis

Binding of hippuric acid (HA), a uremic toxin, with human serum albumin (HSA) has been examined by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), molecular docking, circular dichroism (CD) and fluorescence spectroscopy to understand the reason that govern its impaired elimination through hemodialysis. ITC results shows that the HA binds with HSA at high (K _b ∼10⁴) and low affinity (K _b ∼10³) sites whereas spectroscopic results predict binding at a single site (K _b∼10³). The HA form complex with HSA that involves electrostatic, hydrogen and hydrophobic binding forces as illustrated by calculated thermodynamic parameters. Molecular docking and displacement studies collectively revealed that HA bound to both site I and site II; however, relatively strongly to the later. Esterase-like activity of HSA confirms the involvement of Arg410 and Tyr411 of Sudlow site II in binding of HA. CD results show slight conformational changes occurs in the protein upon ligation that may be responsible for the discrepancy in van’t Hoff and calorimetric enthalpy change. Furthermore, an increase in and is observed from DSC results that indicate increase in stability of HSA upon binding to HA. The combined results provide that HA binds to HSA and thus its elimination is hindered.     

Specific interaction to PIP2 increases the kinetic rate of membrane binding of VILIPs,a subfamily of Neuronal Calcium Sensors (NCS) proteins

VIsinin-LIke Proteins (VILIPs) are a subfamily of the Neuronal Calcium Sensor (NCS) proteins, which possess both N-myristoylation and EF-hand motifs allowing for a putative ‘calcium–myristoyl switch’ regulation mechanism. It has previously been established that myristoyl conjugation increases the affinity of proteins for membranes, but, in many cases, a second feature such as a cluster of positively-charged residues is needed for stable membrane binding. The interaction of two members of this family, VILIP-1 and VILIP-3, with Langmuir monolayers as membrane models has been investigated in order to study the effects of both myristoylation and the highly basic region containing conserved poly-lysine residues on membrane association kinetics and binding properties. Results show that in the presence of calcium, N-myristoylation significantly increases the kinetic rate of VILIP adsorption to the membrane. Additionally, the proteins bind to negatively charged phospholipids independently of the conjugated myristate moiety. Besides the regulatory effect of calcium on the rate of binding presumably due to exposure of the myristoyl moiety ascribed to their putative ‘calcium–myristoyl switch’, VILIP-1 and -3 also engage specific interactions with biomimetic membranes containing phosphatidylinositol 4,5-bisphosphate (PIP₂). The presence of PIP₂ increases the membrane association rates of both VILIPs. Taken together, these results show the major kinetic role of N-myristoylation for membrane binding, and highlight the critical role of specific phosphoinositide interactions for membrane association of members of the VILIP family.     

An evaluation of phycoremediation potential of cyanobacterium Nostoc muscorum: characterization of heavy metal removal efficiency

The present study relates to the use of cyanobacterium Nostoc muscorum as a model system for removal of heavy metals such as Pb and Cd from aquatic systems. The effects of various physicochemical factors on the surface binding and intracellular uptake of Pb and Cd were studied to optimize the metal removal efficiency of the living cells of N. muscorum. Results demonstrated that a significant proportion of Pb and Cd removal was mediated by surface binding of metals (85 % Pb and 79 % Cd), rather than by intracellular accumulation (5 % Pb and 4 % Cd) at the optimum level of cyanobacterial biomass (2.8 g L^?1), metal concentration (80 μg mL^?1), pH (pH 5.0–6.0), time (15–30 min), and temperature (30–40 °C). N. muscorum has maximum amounts of metal removal (q _max) capacity of 833 and 666.7 mg g^?1 protein for Pb and Cd, respectively. The kinetic parameters of metal binding revealed that adsorption of Pb and Cd by N. muscorum followed pseudo-second-order kinetics, and the adsorption behavior was better explained by both Langmuir and Freundlich isotherm models. The surface binding of both the metals was apparently facilitated by the carboxylic, hydroxyl, and amino groups as evident from Fourier transform infrared spectra.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2alpha receptors.

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Binding of a Naja naja venom acidic phospholipase A2 cognate complex to membrane-bound vimentin of rat L6 cells: Implications in cobra venom-induced cytotoxicity

An acidic phospholipase A₂ enzyme (NnPLA₂-I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA₂-I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA₂-I cognate complex to vimentin as compared to the binding of individual NnPLA₂-I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA₂-I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA₂-I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60–240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19–22 residues, inclusive of Asp48 residue, of NnPLA₂-I preferentially binds with the rod domain (99–189 and 261–335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (K_D) values of ?12.86 kcal/mol and 3.67 × 10^?10 M, respectively; however, NnPLA₂-I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity.     

Fluorescent Trimeric Hemagglutinins Reveal Multivalent Receptor Binding Properties

Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA–receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA–sfGFP proteins was studied using glycan arrays and tissue staining. The HA–sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA–sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.     

Electrogenic transport properties of bacteriorhodopsin containing chemically modified retinal analogues

The electrical activity of bacteriorhodopsin (BR) containing the 13-substituted retinal analogues 13-demethyl and 13-methoxy as well as the naturally occurring retinal carrying a methyl group at C13 is compared. White membrane patches reconstituted with the different retinals are attached to a black lipid membrane, and the dependency of the photocurrent on light intensity is measured. This allows a comparison of the overall photocycle time and the number of protons transported per cycle for the various preparations. From previous work (Gärtner, W., Towner, P., Hopf, H. and Oesterhelt, D. (1983) Biochem. 22, 2637–2644, see also Gärtner, W. and Oesterhelt, D., unpublished data) the equilibrium isomeric distribution (all-trans and 13-cis) of the different retinals in the binding site is known. Taking into account that only all-trans retinal BR contributes to the pumping activity (Fahr, A. and Bamberg, E. (1982) FEBS Lett. 140, 251–253), it is shown, that the cycle time for the modified BRs is moderately changed, whereas the number of protons transported per cycle and transporting all-trans BR molecule is not affected by the substituent. It is concluded, that substituting the methyl group at position 13 of the retinal molecule by a hydrogen atom or a methoxy group only slightly affects the pumping activity of the trans-photocycle, but rather controls the biological function of BR via the equilibrium isomeric distribution of the retinal molecule in the binding site.     

Crystal Structure of the HA3 Subcomponent of Clostridium botulinum Type C Progenitor Toxin

The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1 (HA-33), HA2 (HA-17), HA3a (HA-22-23), and HA3b (HA-53). The HA3b subcomponent seems to play an important role cooperatively with HA1 in the internalization of the toxin by gastrointestinal epithelial cells via binding of these subcomponents to specific oligosaccharides. In this study, we investigated the sugar-binding specificity of the HA3b subcomponent using recombinant protein fused to glutathione S-transferase and determined the three-dimensional structure of the HA3a-HA3b complex based on X-ray crystallography. The crystal structure was determined at a resolution of 2.6 Å. HA3b contains three domains, domains I to III, and the structure of domain I resembles HA3a. In crystal packing, three HA3a-HA3b molecules are assembled to form a three-leaved propeller-like structure. The three HA3b domain I and three HA3a alternate, forming a trimer of dimers. In a database search, no proteins with high structural homology to any of the domains (Z score > 10) were found. Especially, HA3a and HA3b domain I, mainly composed of β-sheets, reveal a unique fold. In binding assays, HA3b bound sialic acid with high affinity, but did not bind galactose, N-acetylgalactosamine, or N-acetylglucosamine. The electron density of liganded N-acetylneuraminic acid was determined by crystal soaking. In the sugar-complex structure, the N-acetylneuraminic acid-binding site was located in the cleft formed between domains II and III of HA3b. This report provides the first determination of the three-dimensional structure of the HA3a-HA3b complex and its sialic acid binding site. Our results will provide useful information for elucidating the mechanism of assembly of the C16S toxin and for understanding the interactions with oligosaccharides on epithelial cells and internalization of the botulinum toxin complex.     

Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure–activity relationship studies revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G₂) is a highly selective inhibitor of BR biosynthesis, with an IC₅₀ value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2α receptors

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Roles of Electrostatics and Conformation in Protein-Crystal Interactions

In vitro studies have shown that the phosphoprotein osteopontin (OPN) inhibits the nucleation and growth of hydroxyapatite (HA) and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001). Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions) indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65–80 (pSHDHMDDDDDDDDDGD) and 220–235 (pSHEpSTEQSDAIDpSAEK). In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC₅₀ = 1.93 µg/ml) and OPN220-235 (IC₅₀ = 1.48 µg/ml) are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC₅₀ = 2.97 µg/ml); the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca²⁺-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain.     

Probing the interaction of human serum albumin with bilirubin in the presence of aspirin by multi-spectroscopic, molecular modeling and zeta potential techniques: insight on binary and ternary systems

Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (K(a)) and number of binding site (n) obtained were 1.6 × 10(6)M(-1) and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 10(6)M(-1) and 0.84, respectively, in the presence of ASA (ternary complex) at λ(ex)= 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (C(CIAC)) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.     

Determination of membrane potential with lipophilic cations. Comparison of estimated values with various phosphonium ions

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–5) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the binding of probes to the membrane was measured. For the probes of n = 0 and n = 1, and for TPP⁺, binding followed the Langmuir adsorption isotherm. For other probes, analysis revealed the presence of two, high- and low-affinity, binding sites. Upon illumination, which generated the membrane potential, the probe molecules were accumulated into the vesicles. If we ignore the membrane-potential-dependent binding of the probe molecules, the estimated values are larger when the probe used is more hydrophobic. We have tested some models describing the amount of probe bound on membranes in terms of concentration of free probe inside and outside the vesicles. No model has fulfilled the criterion of valid estimation that the membrane potentials estimated are independent of probes used. An experimental method for the estimation of true membrane potential is proposed. Effects of tetraphenylboron on the estimation of membrane potential and on the transport rate of phosphonium cations were examined.     

Succinate dehydrogenase and fumarate reductase from Escherichia coli

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron–sulfur subunit which contains three distinct iron–sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b₅₅₆ is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

Expression of TWISTED DWARF1 lacking its in‐plane membrane anchor leads to increased cell elongation and hypermorphic growth

Plant growth is achieved predominantly by cellular elongation, which is thought to be controlled on several levels by apoplastic auxin. Auxin export into the apoplast is achieved by plasma membrane efflux catalysts of the PIN‐FORMED (PIN) and ATP‐binding cassette protein subfamily B/phosphor‐glycoprotein (ABCB/PGP) classes; the latter were shown to depend on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Here by using a transgenic approach in combination with phenotypical, biochemical and cell biological analyses we demonstrate the importance of a putative C‐terminal in‐plane membrane anchor of TWD1 in the regulation of ABCB‐mediated auxin transport. In contrast with dwarfed twd1 loss‐of‐function alleles, TWD1 gain‐of‐function lines that lack a putative in‐plane membrane anchor (HA–TWD1‐C_t) show hypermorphic plant architecture, characterized by enhanced stem length and leaf surface but reduced shoot branching. Greater hypocotyl length is the result of enhanced cell elongation that correlates with reduced polar auxin transport capacity for HA–TWD1‐C_t. As a consequence, HA–TWD1‐C_t displays higher hypocotyl auxin accumulation, which is shown to result in elevated auxin‐induced cell elongation rates. Our data highlight the importance of C‐terminal membrane anchoring for TWD1 action, which is required for specific regulation of ABCB‐mediated auxin transport. These data support a model in which TWD1 controls lateral ABCB1‐mediated export into the apoplast, which is required for auxin‐mediated cell elongation.     

Effects of alterations of the amino-terminal glycine of influenza hemagglutinin fusion peptide on its structure, organization and membrane interactions

Mutations of the glycine residue at the amino terminus of HA2 have been shown to have a large effect on the fusion activity of HA2, the extent of which apparently correlates with the side chain bulkiness of the substituting amino acids. To investigate into the cause of abrogation in fusogenicity and virus-promoted fusion mechanism, we synthesized several peptides in which this glycine was substituted by serine, glutamic acid, or lysine. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl sn-glycero-3-phosphoglycerol (DMPG) were used as model membranes in the fluorescence, circular dichroism (CD), and FTIR measurements while sodium dodecyl sulfate was used in NMR studies. We found that, for the less active variants, affinity to membrane, degree of solvent dehydration, lipid perturbation, depth of insertion, and helicity were less. Comparison of affinity to membrane bilayer among these analogs revealed that binding of the fusion peptide is determined largely by the hydrophobic effect. Additionally, the orientation is closer to the membrane normal for the wild-type fusion peptide in the helix form while the inactive analogs inserted more parallel to the membrane surface.     

Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

Histamine was immobilized on Sepharose CL‐6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine‐Sepharose (HA‐S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA‐S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (ΔH_ads, ΔS_ads, and ΔG_ads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt‐buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA‐S was characterized by a less favorable ΔG_ads and an unfavorable ΔS_ads because histamine was covalently attached to Sepharose via a three‐carbon‐chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ΔS_ads was offset by a favorable ΔH_ads, thus exhibiting typical enthalpy‐entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA‐S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010