Lysozyme Binding Research Articles

Proton-coupled protein binding: controlling lysozyme/poly(acrylic acid) interactions with pH.

Rational design of protein-polymer composites and their use, under the influence of the stimulus, for numerous applications requires a clear understanding of protein-polymer interfaces. Here, using poly(acrylic acid) (PAA) and lysozyme as model systems, the binding interactions between these macromolecules were investigated by isothermal titration calorimetry. The binding is proposed to require and be governed by "charge neutralization of the protein/polymer interface" and predicted to depend on solution pH. Calorimetric data show strong exothermic binding of lysozyme to PAA with a molar ΔH and TΔS values of -107 and -95 kcal/mol, respectively, at pH 7 and room temperature. Both ΔH and TΔS decreased linearly with increasing pH from 3 to 8, and these plots had slopes of -17.7 and -17.5 kcal/mol per pH unit, respectively. The net result was that the binding propensity (ΔG) was nearly independent of pH but the binding stoichiometry, surprisingly, increased rapidly with increasing pH from 1 lysozyme binding per PAA molecule at pH 3 to 16 lysozyme molecules binding per PAA molecule at pH 8. A plot of stoichiometry vs pH was linear, and consistent with this result, a plot of ln(average size of the protein/polymer complex) vs pH was also linear. Thus, protonation-deprotonation plays a major role in the binding mechanism. "Charge neutralization" of the lysozyme/PAA interface controls the binding stoichiometry as well as the binding enthalpies/entropies in a predictable fashion, but it did not control the binding affinity (ΔG). The pH dependence of lysozyme binding to PAA, demonstrated here, provides a stimuli-responsive system for protein binding and release from the polymer surface.

Non-covalent conjugation of CdTe QDs with lysozyme binding DNA for fluorescent sensing of lysozyme in complex biological sample

Water-soluble cysteamine (CA) capped CdTe quantum dots (QDs) conjugated with lysozyme binding DNA (LBD) was constructed for luminescent sensing of lysozyme by forming a ternary self-assembly complex. Addition of negatively charged lysozyme binding DNA to the positively charged CA capped CdTe QDs buffer solution (Tris–HCl pH 7.4) could lead to the formation of QDs–LBD complex through electrostatic interactions. Once lysozyme was introduced into the CdTe QDs–LBD system, it could bind specifically with the QDs–LBD complex, resulting in fluorescence emission enhancement of the QDs due to the surface inert of QDs. At a given amount of LBD and CdTe QDs (LBD: QDs=2: 1), the fluorescence intensity enhancement of QDs was linear with lysozyme concentration over the range of 8.9–71.2nM, with a detection limit of 4.3nM. Due to the specific binding of LBD with lysozyme, this approach displayed high selectivity for lysozyme recognition. The sensing mechanism was confirmed by DLS and zeta potential measurement, and agarose gel electrophoresis experiment. Furthermore, the proposed CA–capped CdTe QDs–LBD sensor was applied to lysozyme detection in mouse serum and human morning urine samples, which showed high sensitivity and selectivity in the complex biological sample.

Lysozyme binding to tethered bilayer lipid membranes prepared by rapid solvent exchange and vesicle fusion methods

Tethered bilayer lipid membranes (tBLMs) are important tools for studying protein-lipid interactions. The widely used methodology for the preparation of these membranes is the fusion of phospholipid vesicles from an aqueous medium onto an anchored phospholipid layer. The preparation of phospholipid vesicles is a long and tedious procedure. There is another simple method, rapid solvent exchange, for preparing lipid membranes. However, there is a lack of information on the effects of the preparation method of tBLMs on their interactions with proteins. Therefore, we present in this paper a comparative study on the binding of lysozyme onto tBLMs prepared by the abovementioned methods. The prepared tBLMs have either zwitterionic or anionic characteristics. The results show that lysozyme binding onto the prepared tBLMs is unaffected by the preparation method of the tBLMs, suggesting that the tedious fusion method might be replaced by the simple rapid solvent exchange method without altering the level of protein-lipid interactions.

Understanding the poor iontophoretic transport of lysozyme across the skin: When high charge and high electrophoretic mobility are not enough

The original aim of the study was to investigate the transdermal iontophoretic delivery of lysozyme and to gain further insight into the factors controlling protein electrotransport. Initial experiments were done using porcine skin. Lysozyme transport was quantified by using an activity assay based on the lysis of Micrococcus lysodeikticus and was corrected for the release of endogenous enzyme from the skin during current application. Cumulative iontophoretic permeation of lysozyme during 8h at 0.5mA/cm2 (0.7mM; pH6) was surprisingly low (5.37±3.46μg/cm2 in 8h) as compared to electrotransport of cytochrome c (Cyt c) and ribonuclease A (RNase A) under similar conditions (923.0±496.1 and 170.71±92.13μg/cm2, respectively) — despite its having a higher electrophoretic mobility. The focus of the study then became to understand and explain the causes of its poor iontophoretic transport. Lowering formulation pH to 5 increased histidine protonation in the protein and decreased the ionisation of fixed negative charges in the skin (pI ~4.5) and resulted in a small but statistically significant increase in permeation. Co-iontophoresis of acetaminophen revealed a significant inhibition of electroosmosis; inhibition factors of 12–16 were indicative of strong lysozyme binding to skin. Intriguingly, lidocaine electrotransport, which is due almost exclusively to electromigration, was also decreased (approximately 2.7-fold) following skin pre-treatment by lysozyme iontophoresis (cf. iontophoresis of buffer solution) — suggesting that lysozyme was also able to influence subsequent cation electromigration. In order to elucidate the site of skin binding, different porcine skin models were tested (dermatomed skin with thicknesses of 250 and 750μm, tape-stripped skin and heat-separated dermis). Although no difference was seen between permeation across 250 and 750μm dermatomed skin (13.57±12.20 and 5.37±3.46μg/cm2, respectively), there was a statistically significant increase across tape-stripped skin and heat-separated dermis (36.86±7.48 and 43.42±13.11μg/cm2, respectively) — although transport was still much less than that seen across intact skin for Cyt c or RNase A. Furthermore, electroosmotic inhibition factors fell to 2.2 and 1.0 for tape-stripped skin and heat-separated dermis — indicating that lysozyme affected convective solvent flow through interactions with the epidermis and predominantly the stratum corneum. Finally, cation exchange and hydrophobic interaction chromatography confirmed that although lysozyme had greater positive charge than Cyt c or RNase A under the conditions used for iontophoresis, it also possessed the highest surface hydrophobicity, which may have facilitated the interactions with the transport pathways and encouraged aggregation in the skin microenvironment. Thus, high charge and electrophoretic mobility seem to be inadequate descriptors to predict the transdermal iontophoretic transport of proteins whose complex three dimensional structures can facilitate interactions with cutaneous transport pathways.

Multiscale evaluation of pore curvature effects on protein structure in nanopores.

Protein structure in nanopores is an important determinant in porous substrate utilization in biotechnology and materials science. To date, accurate residue details of pore curvature induced protein binding and unfolding were still unknown. Here, a multiscale ensemble of chromatography, NMR hydrogen and deuterium (H/D) exchange, confocal scanning and molecular docking simulations was combined to obtain the protein adsorption information induced by pore size and curvature. Lysozyme and polystyrene microspheres within pores in the 14-120 nm range were utilized as models. With pore size increasing, the bound lysozyme presented a tendency of significantly decreased retention, less unfolding and fewer interacted sites. However, such a significant dependence between pore curvature and protein size only existed in a limited micro-pore range comparable to protein sizes. The mechanism behind the above events could be attributed to the diverse protein interaction area determined by pore curvature and size change, by models calculating the binding of lysozyme onto surfaces. Another surface of opposite curvature for nanoparticles was also calculated and compared, the rules were similar but with opposite direction and such a critical size also existed. These studies of proteins on curved interfaces may ultimately help to guide the design of novel porous materials and assist in the discrimination of the target protein from molecular banks.

Tuning the Activities and Structures of Enzymes Bound to Graphene Oxide with a Protein Glue

Graphene oxide (GO) is being investigated extensively for enzyme and protein binding, but many enzymes bound to GO denature considerably and lose most of their activities. A simple, novel, and efficient approach is described here for improving the structures and activities of enzymes bound to GO such that bound enzymes are nearly as active as those of the corresponding unbound enzymes. Our strategy is to preadsorb highly cationized bovine serum albumin (cBSA) to passivate GO, and cBSA/GO (bGO) served as an excellent platform for enzyme binding. The binding of met-hemoglobin, glucose oxidase, horseradish peroxidase, BSA, catalase, lysozyme, and cytochrome c indicated improved binding, structure retention, and activities. Nearly 100% of native-like structures of all the seven proteins/enzymes were noted at near monolayer formation of cBSA on GO (400% w/w), and all bound enzymes indicated 100% retention of their activities. A facile, benign, simple, and general method has been developed for the biofunctionalization of GO, and this approach of coating with suitable protein glues expands the utility of GO as an advanced biophilic nanomaterial for applications in catalysis, sensing, and biomedicine.

Measuring Protein–Ligand Interactions Using Liquid Sample Desorption Electrospray Ionization Mass Spectrometry

We have previously shown that liquid sample desorption electrospray ionization-mass spectrometry (DESI-MS) is able to measure large proteins and noncovalently bound protein complexes (to 150 kDa) (Ferguson et al., Anal. Chem. 2011, 83, 6468-6473). In this study, we further investigate the application of liquid sample DESI-MS to probe protein-ligand interactions. Liquid sample DESI allows the direct formation of intact protein-ligand complex ions by spraying ligands toward separate protein sample solutions. This type of "reactive" DESI methodology can provide rapid information on binding stiochiometry, selectivity, and kinetics, as demonstrated by the binding of ribonuclease A (RNaseA, 13.7 kDa) with cytidine nucleotide ligands and the binding of lysozyme (14.3 kDa) with acetyl chitose ligands. A higher throughput method for ligand screening by liquid sample DESI was demonstrated, in which different ligands were sequentially injected as a segmented flow for DESI ionization. Furthermore, supercharging to enhance analyte charge can be integrated with liquid sample DESI-MS, without interfering with the formation of protein-ligand complexes.

Protein Composition of Different Sized Casein Micelles in Milk after the Binding of Lactoferrin or Lysozyme

Casein micelles with bound lactoferrin or lysozyme were fractionated into sizes ranging in radius from ∼50 to 100 nm. The κ-casein content decreased markedly and the αS-casein/β-casein content increased slightly as micelle size increased. For lactoferrin, higher levels were bound to smaller micelles. The lactoferrin/κ-casein ratio was constant for all micelle sizes, whereas the lactoferrin/αS-casein and lactoferrin/β-casein ratio decreased with increasing micelle size. This indicates that the lactoferrin was binding to the surface of the casein micelles. For lysozyme, higher levels bound to larger casein micelles. The lysozyme/αS-casein and lysozyme/β-casein ratios were nearly constant, whereas the lysozyme/κ-casein ratio increased with increasing micelle size, indicating that lysozyme bound to αS-casein and β-casein in the micelle core. Lactoferrin is a large protein that cannot enter the casein protein mesh; therefore, it binds to the micelle surface. The smaller lysozyme can enter the protein mesh and therefore binds to the more charged αS-casein and β-casein.

Overdamped Dynamics of Folded Protein Domains within a Locally Harmonic Basin Using Coarse Graining Based on a Partition of Compact Flexible Clusters

A coarse-graining method based on the partitioning of atoms into compact flexible clusters is used to formulate the dynamics of the nonequilibrium response of a protein to ligand dissociation. The α-carbon positions are used as the degrees of freedom. The net stiffness between each pair of neighboring α-carbons is calculated for the quasi-static, overdamped regime within the harmonic (quadratic potential energy surface) using the equivalent stiffness matrix of the network of atoms occupying the intervening space within the locally interacting region. This localized approach realizes a divide and conquer strategy that results in a substantial reduction in computational complexity while accurately predicting relaxations under general loading conditions. A close correlation between the shapes and time scales of the relaxation curves of the coarse-grained and all-atom instances of two medium-sized proteins, T4 lysozyme and ferric binding protein (each of which having known apo and holo structures), was observed for the holo to the apo transitions. Furthermore, for both proteins the dominant modes of motion and the decay rates of the temporal relaxation profiles monitoring the separation distance between select amino acid pairs were found to be nearly identical when calculated on the coarse-grained and all-atom scales.

Graphene-based lysozyme binding aptamer nanocomposite for label-free and sensitive lysozyme sensing

This work presents a facile route to fabricate a highly sensitive platform for the recognition of lysozyme. Graphene oxide was coated on the surface of a glassy carbon electrode (GCE) followed by reduction with hydrazine to obtain a graphene (GR) coated GCE. After activation of GR–GCE with EDC and NHS, lysozyme binding aptamer (LBA) was molecularly tethered onto GR to obtain a highly sensitive platform named as LBA–GR–GCE. Results show that GR greatly promotes the electron transfer at electrode/electrolyte interface due to its excellent electrical conductivity. LBA–GR–GCE is able to quantitatively detect the analyte of lysozyme in real samples of human saliva and chicken egg white with high selectivity, with a linear range of 0.01–0.5pmolL−1 and detection limit of 6fmolL−1 (S/N=3). Furthermore, the surface feature of the functionalized electrode was investigated to determine its effect on charge transfer resistance with two different electrochemical probes of [Fe(CN)6]3−/4− and Ru(NH3)63+.

Colorimetric and fluorometric dual-readout sensor for lysozyme

A novel, highly sensitive and selective dual-readout sensor (colorimetric and fluorometric) for the detection of lysozyme was proposed. The fluorescence of triazolylcoumarin molecules was quenched by Au nanoparticles (AuNPs) initially through the fluorescence resonance energy transfer (FRET), after the addition of lysozyme, the stronger binding of lysozyme onto the surfaces of AuNPs made triazolylcoumarin molecules remove from the AuNPs surface and led to the recovery of the fluorescence of triazolylcoumarin molecules, and accompanied by the discernable color change of the solution from red to purple. The lowest detectable concentration for lysozyme was 50 ng mL(-1) by the naked eye, and the limit of detection (LOD) was 23 ng mL(-1) by fluorescence measurements. In addition, satisfactory results for lysozyme detection in hen egg white were confirmed in the study. Moreover, the presented sensor provides a reliable option to determine lysozyme with high sensitivity and selectivity.

Tuning the selective interaction of lysozyme and serum albumin on a carboxylate modified surface

We have demonstrated an efficacious approach for tuning the selective adsorption of hen egg white lysozyme (HEWL) and bovine serum albumin (BSA) on the same carboxylate modified surface at different pH values. A basic alumina surface has been modified with trimesic acid to generate a carboxylated surface, and it has been used for the first time in differential protein interaction studies. We have found that very simple surface chemistry can be utilized for the controlled and selective adsorption of both proteins as a function of pH, which leads to the preferential binding of lysozyme over BSA at physiological pH ∼ 7.4. Significantly, we achieve a high retention of the enzymatic activity of the adsorbed lysozyme (∼98% of that of the native enzyme) at lower surface coverage, which also persists in different harsh conditions. In-depth conformational analysis revealed that selectively adsorbed lysozyme was partially unfolded but mostly retained its secondary structural content. In addition, we have also proposed an explanation of the possible interaction behaviour of the carboxylated surface with BSA and lysozyme molecules with the help of surface potential analysis, which provides an in-depth understanding of differential protein interaction.

The effect of lysozyme amyloid fibrils on cytochrome c–lipid interactions

Protein polymerization into ordered fibrillar structures (amyloid fibrils) is currently associated with a range of pathological conditions. Recent studies clearly indicate that amyloid cytotoxicity is provoked by a continuum of cross-β-sheet aggregates including mature fibrils. In view of the possible diversity of cytotoxicity mechanisms, the present study addressed the question of whether protein conversion into amyloid fibrils can modify its competitive membrane adsorption behavior. Using a combination of resonance energy transfer, dynamic light scattering and fluorescence quenching techniques, the competitive binding of either monomeric or polymerized lysozyme, and cytochrome c to the model lipid membranes composed of phosphatidylcholine mixtures with varying proportions of phosphatidylglycerol, phosphatidylserine or cardiolipin has been studied. The ability of fibrillar lysozyme to induce dissociation of cytochrome c from the membrane binding sites proved to be markedly stronger than that of its monomeric counterpart, with desorption process displaying cooperativity features upon increasing the charge of lipid bilayer. The decreased efficiency of tryptophan fluorescence quenching by acrylamide and short-wavelength shift of emission maximum observed upon membrane binding of lysozyme fibrils were rationalized in terms of fluorophore transfer into interfacial bilayer region. It is hypothesized that electrostatic interactions play predominant role in determining the lipid-associating and competitive abilities of fibrillar lysozyme.

The Lysozyme-Induced Peptidoglycan N -Acetylglucosamine Deacetylase PgdA (EF1843) Is Required for Enterococcus faecalis Virulence

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O-acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.

Determination of Picogram Amounts of Dihydroxybenzenes in Water Samples with Luminol–lysozyme Chemiluminescence System

AbstractAn ultrasensitive method for the determination of dihydroxybenzens by flow injection (FI) chemiluminescence (CL) analysis was proposed for the first time. It was found that the CL intensity of luminol–lysozyme system could be significantly inhibited by dihydroxybenzens. The CL intensity decrements were linear with the logarithm of dihydroxybenzens concentrations over the ranges of 1.0 ∼ 700 pg mL‐1 for hydroquinone (HQ), 5.0 ∼ 700 pg mL‐1 for catechol (CT) and 10 ∼ 7000 pg mL‐1 for resorcinol (RS), with the corresponding limits of detection of 0.7, 3.0 and 7.0 pg mL‐1, respectively. The proposed method was successfully applied to the determination of CT in tap water, rain water, river water and HQ in waste photographic developer samples, with recoveries from 93.5 to 105.8% and relative standard deviations (RSDs) less than 4.0% (n = 5). The possible interaction mechanism of lysozyme with dihydroxybenzens was discussed, and CT to lysozyme's binding constant and the thermodynamic parameters were given by the homemade FI–CL model. The results shown that the binding of dihydroxybenzens to lysozyme was spontaneous with the hydrophobic force.

A three-way junction aptasensor for lysozyme detection

A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called “conductive path”, is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2nM with a dynamic range extending to 100nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure.

Molecularly imprinted stimuli-responsive hydrogels for protein recognition

Temperature-responsive poly(N-isopropylacrylamide)-based (PNIPAAm) hydrogels were imprinted with lysozyme via in situ photo-initiated crosslinking polymerization. The three-dimensional network of the hydrogels was tailored by tuning the ionic content through methacrylic acid as template-binding comonomer while keeping the ratio between crosslinker (N,N′-methylenebisacrylamide) and N-isopropylacrylamide fixed. Moderate salt concentrations (0.3 m NaCl) were found to be suited for template removal without phase separation of the hydrogel. Swelling and protein (lysozyme and cytochrome C) binding were investigated for imprinted and nonimprinted gels at temperatures below and above the lower critical solution temperature of PNIPAAm (32 °C). Imprinted gels showed a much higher affinity, selectivity and binding capacity for lysozyme compared to the nonimprinted reference materials. Protein binding capacity was strongly reduced above 32 °C, to zero for nonimprinted and to small values for imprinted gels. Most important, specific lysozyme binding to the imprinted gels caused a large concentration dependent deswelling. This effect was much smaller for nonimprinted gels, and the response could be modulated by the content of the comonomer methacrylic acid. Overall, this approach is interesting for the development of novel sensors or materials for controlled release applications.

The dynamical response of hen egg white lysozyme to the binding of a carbohydrate ligand

It has become clear that the binding of small and large ligands to proteins can invoke significant changes in side chain and main chain motion in the fast picosecond to nanosecond timescale. Recently, the use of a "dynamical proxy" has indicated that changes in these motions often reflect significant changes in conformational entropy. These entropic contributions are sometimes of the same order as the total entropy of binding. Thus, it is important to understand the connections amongst motion between the manifold of states accessible to the native state of proteins, the corresponding entropy, and how this impacts the overall energetics of protein function. The interaction of proteins with carbohydrate ligands is central to a range of biological functions. Here, we examine a classic carbohydrate interaction with an enzyme: the binding of wild-type hen egg white lysozyme (HEWL) to the natural, competitive inhibitor chitotriose. Using NMR relaxation experiments, backbone amide and side chain methyl axial order parameters were obtained across apo and chitotriose-bound HEWL. Upon binding, changes in the apparent amplitude of picosecond to nanosecond main chain and side chain motions are seen across the protein. Indeed, binding of chitotriose renders a large contiguous fraction of HEWL effectively completely rigid. Changes in methyl flexibility are most pronounced closest to the binding site, but average to only a small overall change in the dynamics across the protein. The corresponding change in conformational entropy is unfavorable and estimated to be a significant fraction of the total binding entropy.

Porous silicon biosensor for detection of variable domain of heavy-chain of HCAb antibody

In this paper, we produce porous silicon (PSi) by electrochemical etching, and it is the first time to evaluate the performance of label-free porous silicon biosensor for detection of variable domain of heavy chain of heavy-chain antibody (VHH). The binding of hen egg white lysozyme (HEWL) and VHH causes a red shift in the reflection spectrum of the biosensor. The red shift is proportional to the VHH concentration in the range from 14 g·ml−1 to 30 g·ml−1 with a detection limit of 0.648 ng·ml−1. The research is useful for the development of label-free biosensor applied in the rapid and sensitive determination of small molecules.

Newly folded substrates inside the molecular cage of the HtrA chaperone DegQ

The HtrA protein family combines chaperone and protease activities and is essential for protein quality control in many organisms. Whereas the mechanisms underlying the proteolytic function of HtrA proteins have been analyzed in detail, their chaperone activity remains poorly characterized. Here we describe cryo-electron microscopic structures of Escherichia coli DegQ in its 12- and 24-mer states in complex with model substrates, providing a structural model of HtrA proteins in their chaperone mode. Up to six lysozyme substrates bind inside the DegQ 12-mer cage and are visualized in a close-to-native state. An asymmetric reconstruction reveals the binding of a well-ordered lysozyme to four DegQ protomers. DegQ PDZ domains are located adjacent to substrate density and their presence is required for chaperone activity. The substrate-interacting regions appear conserved in 12- and 24-mer cages, suggesting a common mechanism of chaperone function.