Model Protein System Research Articles

The interaction of disulfide flavor compounds with proteins in model systems.

Sulfur-containing compounds are extremely important in cooked foods for roast, savory, meaty, and coffee-like aromas and flavors (Gasser and Grosch, 1988; Mottram et al., 1998). Many have low odor threshold values and, therefore, relatively small quantities in a food product can have a major influence on the aroma. Recently it has been shown that irreversible binding of such compounds to food proteins can occur, with a possible change in flavor perception of the food. This has implications for consumer acceptance of certain food products where these compounds are used as flavorings or where they occur naturally.KeywordsSulfhydryl GroupWater BlankInterchange ReactionFlavor PerceptionThiolate AnionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Comprehensive study of acid gelation of heated milk with model protein systems

Abstract The effect of heat treatment of milk on the formation of acid gels was investigated using model protein systems. Protein systems contained micellar casein alone (35 g kg −1 ) (systR) or in the combination with either β -lactoglobulin ( β LG) (4 g kg −1 ; systB) or egg ovalbumin (4 g kg −1 ; systO). Proteins in a milk ultrafiltrate (UF) were heat-treated at 90°C for 24 min. The same heat-treated systems without casein were also prepared: the UF alone (systUFR), with β LG (4 g kg −1 ; systUFB) and with ovalbumin (4 g kg −1 ; systUFO). Proteins in pellet and supernatant fractions were analysed using reverse-phase high-pressure liquid chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The solubility of globular proteins in heated systUFB and heated systUFO was determined at pH 1.6–6.5 by absorbance measurements. Rheological changes during gelation with a starter at 42°C were determined and microstructures of gels by scanning electron microscopy. High quantities of κ -casein and β LG in the form of complexes (disrupted by 2-mercaptoethanol) were found in supernatant of heated systB and not in that of systO. The gelation pH was related to the solubility of the globular proteins, and the gelation pattern for systB and systO resembled that of heated milk and the systR pattern resembled that of unheated milk. The gels of systR were coarse and consisted of large particles. Particles in gels from systB and systO were smaller and less clustered. Although the two globular proteins behave differently during heating, they both confer to the casein micelles their solubility properties as a function of pH. A model is presented where both soluble and colloidal complexes including the globular protein interact at pH 5.5 and initiate the acid gelation.

Perturbation of Protein Tertiary Structure in Frozen Solutions Revealed by 1-Anilino-8-Naphthalene Sulfonate Fluorescence

Although freeze-induced perturbations of the protein native fold are common, the underlying mechanism is poorly understood owing to the difficulty of monitoring their structure in ice. In this report we propose that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a useful monitor of ice-induced strains on the native fold. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa, as a model protein system, demonstrate that in frozen solutions the fluorescence of ANS is enhanced several fold and becomes blue shifted relative free ANS. From the enhancement factor it is estimated that, at −13°C, on average at least 1.6 ANS molecules become immobilized within hydrophobic sites of apo-azurin, sites that are destroyed when the structure is largely unfolded by guanidinium hydrochloride. The extent of ANS binding is influenced by temperature of ice as well as by conditions that affect the stability of the globular structure. Lowering the temperature from −4°C to −18°C leads to an apparent increase in the number of binding sites, an indication that low temperature and /or a reduced amount of liquid water augment the strain on the protein tertiary structure. It is significant that ANS binding is practically abolished when the native fold is stabilized upon formation of the Cd 2+ complex or on addition of glycerol to the solution but is further enhanced in the presence of NaSCN, a known destabilizing agent. The results of the present study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.

Protein characterization using Liquid Chromatography Desorption Ionization on Silicon Mass Spectrometry (LC‐DIOS‐MS)

This paper presents a novel combination of of‐line liquid chromatography (LC) separation with desorption ionization on silicon mass spectrometry (DIOS‐MS) as an alternative to the traditional on‐line coupling of LC and electrospray ionization (ESI). In this work, electrospray deposition is used to generate a spatially preserved linear track of the separated sample on a specially designed DIOS chip. The total sequence coverage analysis of two model protein systems was evaluated by both LC‐DIOS‐MS and nanoLC ESI tandem MS (MS/MS). LC‐DIOS‐MS yielded improved sequence coverage for both of the model systems (between 99.5 and 100%) compared with traditional LC‐ESI‐MS/MS analysis (between 82 and 87.6%). In addition to improved sequence coverage determination, LC‐DIOS‐MS also offers the potential for high‐throughput protein characterization.

FAMSBASE: Modeling Database of 161 Genomes

We had participated in the CASP (Community Wide Experiment on the Critical Assessment of Tech-niques for Protein Structure Prediction) that is the competition of the protein structure predictionin 2000 and 2002. Our server were estimated almost the best as the individual homology modelingserver in the CAFASP3 (Critical Assessment of Fully Automated Structure Prediction) that is thefull automatic homology modeling section of CASP5. In this competition we used homology modelingsoftware FAMS [1] (Full automatic protein modeling system) that we had been developed for severalyears [3]. Thus, usefulness of the software FAMS was clearly shown in these competitions.We also have developed FAMSBASE that is web interface of FAMS model structure database. Inthis article we describe that proteins coded in the 161 species genomes were modeled.The commercial version of FAMS modeling software service is available in In-Silico Sciences,Inc. (http://www.pd-fams.com). FAMSBASE is available in http://famsbase.bio.nagoya-u.ac.jp/[4] or http://www.pdfams.jbic.or.jp/.

Heteronuclear multidimensional NMR spectroscopy of solubilized membrane proteins: resonance assignment of native bacteriorhodopsin.

Unraveling the loops: Use of recently developed NMR spectroscopy techniques can provide information about the loop regions that connect the transmembrane helices of membrane proteins such as G-protein-coupled receptors. A solution-state NMR study of the native integral membrane protein bacteriorhodopsin is presented. Bacteriorhodopsin was used as a model system for proteins that consist of seven transmembrane helices. Experiments applied TROSY-type NMR spectroscopy techniques to uniformly 2H/15N- and 2H/13C/15N-labeled samples solubilized with dodecylmaltoside. The assignment of 81 out of 248 residues, mainly in the loop regions, is given (see figure). Structural and dynamic information is derived from the data. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2002/z380_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Structure of a de novo designed protein model of radical enzymes.

The use of side chains as catalytic cofactors for protein mediated redox chemistry raises significant mechanistic issues as to how these amino acids are activated toward radical chemistry in a controlled manner. De novo protein design has been used to examine the structural basis for the creation and maintenance of a tryptophanyl radical in a three-helix bundle protein maquette. Here we report the detailed structural analysis of the protein by multidimensional NMR methods. An interesting feature of the structure is an apparent pi-cation interaction involving the sole tryptophan and a lysine side chain. Hybrid density functional calculations support the notion that this interaction raises the reduction potential of the W degrees /WH redox pair and helps explain the redox characteristics of the protein. This model protein system therefore provides a powerful model for exploring the structural basis for controlled radical chemistry in protein.

Mechanisms of Interaction Between Vanillin and Milk Proteins in Model Systems

ABSTRACT: The effects of milk proteins on vanillin intensity reduction were studied in a phosphate buffer (pH 6.5) using urea and sodium dodecyl sulfate (SDS) as bond disrupting agents. The reduction of vanillin occurred immediately as the protein was introduced to the system. Bovine serum albumin (BSA) interacted more with vanillin than sodium caseinate (CAS) did. Heat treatment had no effect on vanillin reduction of the CAS system; however, free vanillin content was higher in the heated than nonheated BSA system. Hydrogen bonding appeared to be a major force for the interaction of vanillin and CAS. However, hydrophobic interaction seemed to be more important than hydrogen bonding in the vanillin and BSA system.

High pressure-induced changes of biological membrane. Study on the membrane-bound Na(+)/K(+)-ATPase as a model system.

In order to study the pressure-induced changes of biological membrane, hydrostatic pressures of from 0.1 to 400 MPa were applied to membrane-bound Na(+)/K(+)-ATPase from pig kidney as a model system of protein and lipid membrane. The activity showed at least a three-step change induced by pressures of 0.1-100 MPa, 100-220 MPa, and 220 MPa or higher. At pressures of 100 MPa or lower a decrease in the fluidity of lipid bilayer and a reversible conformational change in transmembrane protein is induced, leading to the functional disorder of membrane-associated ATPase activity. A pressure of 100-220 MPa causes a reversible phase transition in parts of the lipid bilayer from the liquid crystalline to the gel phase and the dissociation of and/or conformational changes in the protein subunits. These changes could cause a separation of the interface between alpha and beta subunits and between protein and the lipid bilayer to create transmembrane tunnels at the interface. Tunnels would be filled with water from the aqueous environment and take up tritiated water. A pressure of 220 MPa or higher irreversibly destroys and fragments the gross membrane structure, due to protein unfolding and interface separation, which is amplified by the increased pressure. These findings provide an explanation for the high pressure-induced membrane-damage to subcellular organelles.

Luminescent Quantum Dot-Adaptor Protein-Antibody Conjugates for Use in Fluoroimmunoassays

A method for the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe–ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays is presented. The conjugation strategy employs two routes: 1. Use of an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies, and 2. use of avidin, also electrostatically self-assembled onto the nanocrystal surface, which allows QD conjugation to biotinylated antibodies via avidin–biotin binding scheme. With this approach, the average number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed composition conjugates of the molecular adaptor and a second “inert” two-domain fusion protein that allows the use of affinity chromatography. QD/adaptor-antibody conjugates were successfully employed in fluoroimmunoassays for the detection of small molecule analytes, 2,4,6-trinitrobenzene (TNB) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We also demonstrate the use of QD/avidin-antibody conjugates for fluoroimmunoassays using a model protein system.

FAMS and FAMSBASE: Modeling of All the Genomes and Database

We had participated in the CASP4 (Fourth Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction) that is the competition of the protein structure prediction in 2000 [3]. Our server were estimated almost the best in the CAFASP2 (Critical Assessment of Fully Automated Structure Prediction) that is the full automatic homology modeling section of CASP4 [1, 2]. In this competition we used homology modeling software FAMS (Full automatic protein modeling system) that we had been developed for several years [4]. Therefore usefulness of the software FAMS was clearly shown in these competitions. We also have developed software FAMSBASE that is web browser interface of FAMS model structure database. In this article we describe that proteins coded in the all the genomes were modeled. The FAMS modeling web service is available in http://physchem.pharm.kitasato-u.ac.jp/. FAMSBASE is available in http://prtds.pharm.kitasato-u.ac.jp/ or http://famsbase.bio. nagoya-u.ac.jp/.

Characterization of soluble amaranth and soybean proteins based on fluorescence, hydrophobicity, electrophoresis, amino Acid analysis, circular dichroism, and differential scanning calorimetry measurements.

Intrinsic fluorescence (IF), surface hydrophobicity (S(o)), electrophoresis, amino acid analysis, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study folded and unfolded soluble proteins from Amaranthus hypochondriacus (A. h.) and soybean (S). Globulin (Glo) and albumin subfractions (Alb-1 and Alb-2) were extracted from A. h. and S and denatured with urea. Electrophoretic and functional properties indicated a significant correlation between soluble protein fractions from soybean and amaranth. The protein fractions shared some common electrophoretic bands as well as a similar amino acid composition. The larger percent of denaturation in protein fractions, which is associated with enthalpy and the number of ruptured hydrogen bonds, corresponds to disappearance of alpha-helix. The obtained results provided evidence of differences in their secondary and tertiary structures. The most stable was Glo followed by the Alb-2 fraction. Predicted functional changes in model protein systems such as pseudocereals and legumes in response to processing conditions may be encountered in pharmaceutical and food industries. These plants can be a substitute for some cereals.

Structure of the macromolecular solutions that generate crystals

Biomolecular interactions in solution include a variety of effects: hard sphere, electrostatic, van der Waals, hydrophobic, etc. The corresponding interaction potentials govern the macromolecular distribution in solution, the shapes of the phase diagrams and the crystallization process. Different techniques: small angle X-ray scattering (SAXS), light scattering, osmotic pressure, can be used to characterize these potentials. Here, SAXS was the tool of choice to follow the changes induced by the crystallizing agents in different physicochemical conditions. Moreover, the coupling of SAXS (experimental structure factors) and numerical simulations derived from statistical mechanics (calculated structure factors) allowed us to determine the best fit parameters of the relevant potentials. Several protein model systems have now been investigated, with different isoelectric points, sizes and compactness. After the studies performed on lysozyme, and on aspartate transcarbamylase (ATCase), we proceeded with lens gamma- and alpha-crystallins and with urate oxidase. We can now draw the following picture of the relevant potentials. With low molecular weight proteins, a coulombic, pH dependent, repulsive potential and a short range (a few Å), possibly van der Waals, attraction are sufficient to account for the behavior observed at low ionic strength. At higher ionic strength, the salt specific effects that follow the (direct or reverse) order of the Hofmeister series, correspond to an additional short range salt specific attraction. With increasing protein size, the van der Waals contribution becomes negligible. The addition of polymers like polyethylene glycol (PEG) induces a depletion mechanism, which is equivalent to a protein–protein attraction. Crystallization definitively appears to be under control of sufficiently strong and short range (a few Å) attractions, which means that, despite the diversity of protein sequences, it is possible to define a range of physicochemical conditions that may generate crystals. Yet, if salt addition appears to be sufficient to provide such conditions with small compact proteins, additives like PEG seem to be required at higher molecular weights. The success, of course, will also rely upon the protein purity and stability.

Dependence of nucleation kinetics and crystal morphology of a model protein system on ionic strength

Nucleation rate data for hen egg-white lysozyme crystallization were obtained using a particle counter. Tetragonal lysozyme crystals were expected to form at the temperature and solution conditions of these experiments: 4°C, pH 4.5 with 0.1 M sodium acetate buffer and 2–6% NaCl (w/v). The rates varied as expected, as smooth monotonic functions of supersaturation at 2%, 3% and 6% NaCl. However, at 5% NaCl, a great deal of scatter in the data was observed. At 2% and 3% NaCl, all the batches contained crystals with tetragonal morphology. At 6% NaCl, almost all of the vials contained the white powder with few or no tetragonal crystals. At 5% NaCl concentration, a mixture of tetragonal crystals and powder formed in varying proportions in all the vials as observed by visual inspection. The powdery material was examined using optical microscopy and was seen to consist of needles with regular structure and sharp, faceted edges. Powder diffraction data from these needles was inconsistent with experimental powder diffraction data from tetragonal lysozyme crystals. It is possible that at high salt and protein concentrations liquid-liquid separation occurred and yielded a crystal polymorph.

Improving the Sensitivity of a Quartz Crystal Microbalance for Biosensing by Using Porous Gold

A quartz crystal microbalance can be used as a biosensor if biological receptor molecules (ligands) are attached to the crystal. To improve the sensitivity, the surface area of the gold electrodes on the crystal was increased by rendering the electrodes porous. This increased the response up to a factor of 3. A protein model system with human anti-myoglobin as ligand and sheep skeletal myoglobin at different concentrations as analyte was used. The quartz crystal was mounted in a flow-cell, where immobilisation, binding and regeneration of the surface were carried out. The kinetics of the model system was monitored under various experimental conditions.

Measurements of protein stability by H/D exchange and matrix-assisted laser desorption/ionization mass spectrometry using picomoles of material.

Recently, we reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique, termed SUPREX, that can be used to measure the thermodynamic stability of a protein (Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8296-8301). In the work described here, we report on our efforts to optimize the sensitivity of SUPREX analyses. We describe a new sample handling protocol for SUPREX that involves the use of batch chromatography methods with reversed-phase chromatographic media for the microconcentration and desalting of SUPREX samples. Using ribonuclease A as a model protein system, we demonstrate that our new protocol permits the SUPREX analysis of as little as 10 pmol of protein. This amount of protein is 100-fold less than the amount of material required in our initial SUPREX protocol, and it is 1-2 orders of magnitude less than the amount of material required in conventional spectroscopy-based methods for measuring the thermodynamic stability of a protein.

Aggregation in a concentrated model protein system: a mesoscopic simulation of β-casein self-assembly

A mesoscopic model of a concentrated system of amphiphilic co-polymer molecules has been investigated in three dimensions by Brownian dynamics computer simulation. Each co-polymer species consists of a flexible bonded chain of four hydrophilic segments and 12 hydrophobic segments. Equilibrium configurations have been generated as a function of the polymer volume fraction and the strengths of the hydrophobic and electrostatic segment–segment interactions. Some quantitative data are reported on segment co-ordination numbers, maximum aggregate sizes and diffusion coefficients. The mesoscopic simulation approach is put forward as a simple systematic way of modelling a solution of β-casein-like molecules under concentrated conditions. Subject to suitable modifications and refinements, this approach provides a framework for investigating effects of protein concentration and solution conditions on the structure of self-assembled casein systems in food gels and emulsions.

Protein adsorption to oligo(ethylene glycol) self-assembled monolayers: Experiments with fibrinogen, heparinized plasma, and serum

Low protein adsorption is believed advantageous for blood-contacting materials and ethylene glycols (EG)-based polymeric compounds are often attached to surfaces for this purpose. In the present study, the adsorption of fibrinogen, serum, and plasma were studied by ellipsometry on a series of well-defined oligo(EG) terminated alkane-thiols self-assembled on gold. The layers were prepared with compounds of the general structure HS-(CH2)15-CONH-EGn, where n = 2, 4, and 6. Methoxy-terminated tri(EG) undecanethiol and hydroxyl-terminated hexadecanethiol self-assembled monolayers (SAMs) were used as references. The results clearly demonstrate that the adsorption depends on the experimental conditions with small amounts of fibrinogen adsorbing from a single protein solution, but larger amounts of proteins from serum and plasma. The adsorption of fibrinogen and blood plasma decreased with an increasing number of EG repeats and was temperature-dependent. Significantly less serum adsorbed to methoxy tri(EG) than to hexa(EG) and more proteins remained on the latter surface after incubation in a sodium dodecyl sulfate (SDS) solution, indicating a looser protein binding to the methoxy-terminated surface. All surfaces adsorbed complement factor 3 (C3) from serum and plasma, although no surfacemediated complement activation was observed. The present study points to the importance of a careful choice of the protein model system before general statements regarding the protein repellant properties of potential surfaces can be made.

Perturbation of Lipid and Protein Structure by General Anesthetics: How Little Is Too Little?

Assistant Professor of Anesthesia Harvard Medical SchoolAssistant AnesthetistMassachusetts General HospitalDepartment of AnesthesiaBoston, Massachusetts 02114Raines@etherdome.MGH.harvard.edu.To the Editor:—Lipid-based theories of general anesthetic action have long endured because numerous studies have shown that the in vivo pharmacology of an anesthetic correlates remarkably well with its ability to perturb the structural properties of simple lipid bilayers. The Meyer-Overton correlation between anesthetic potency and hydrophobicity, the inactivity of nonanesthetic long chain alcohols and highly halogenated volatile compounds (nonimmobilizers), and pressure reversal have all been demonstrated in studies using protein-free lipid bilayers. 1–6Nevertheless, a most persuasive and often mentioned argument against lipid-based theories is that at clinically relevant concentrations, anesthetics induce only small perturbations in lipid bilayer structure. 7,8For example, halothane reduces the order parameter (increase the “fluidity”) of lipid bilayers by only 1% at clinically relevant concentrations. 9An equivalent reduction in order parameter may be obtained by raising the temperature of the bilayer by less than 1°C. Similarly, halothane reduces the transition temperature between a lipid bilayer’s liquid and gel phases by only 0.5°C at anesthetic concentrations and by only 5°C even at 10 times the minimum aveolar concentration (MAC). 10I was, therefore, very interested to read the study by Johansson et al. quantifying the effects of isoflurane and halothane on structural properties of bovine serum albumin, a lipid-free protein model used in mechanistic studies of anesthetic action. 11What did their studies show? At approximately 1 MAC, isoflurane and halothane increased the fluorescence anisotropy of bovine serum albumin by 1%. An equivalent reduction was obtained by raising the temperature of bovine serum albumin by approximately 1° C. Similarly, isoflurane and halothane raised the transition temperature between the folded and unfolded states of bovine serum albumin by less than 1°C at anesthetic concentrations and by only 3–4°C even at 10 times MAC. Studies of anesthetic binding to other protein models have been similarly unable to demonstrate significant anesthetic-induced changes in protein structure. 12,13Thus, anesthetics induce similarly small changes in the structural properties of lipids and proteins. For consistency, shouldn’t we now conclude that such insensitivity argues strongly against a protein site of anesthetic action?The inability to detect significant anesthetic-induced structural changes in either lipid or protein model systems highlights the practical (and obvious) limitations of such studies: we can only measure what we can measure. Fluorescence anisotropy, denaturation temperature, phase transition temperature, and order parameter have been used by biophysicists for many years as indicators of lipid bilayer and protein structure in large part because they are easily quantitated. There is no compelling theoretical reason to believe that changes in these properties directly accounts for the functional effects of anesthetics on relevant targets in the central nervous system. In fact, it seems quite likely that the anesthetic state results from changes in other lipid and/or protein physical properties that are not so easily measured.

Globular Dendrimers Involving a C60 Core and a Tetraphenyl Porphyrin Function

The nanoenvironment provided by the neighboring dendra characteristically influences the redox potentials and the photophysical properties of the tetraphenylporphyrin–zinc (Zn-TPP; denoted as P in Figure) chromophore within the dendrimers 1, which are based on C60 as a structure-determining core. Such functional dendrimers represent a new prototype of model systems for heme proteins (1).