Non-denatured Protein Research Articles

Nanotoxoid vaccines

To improve innate defense against diseases, vaccine formulations are routinely administered to mount immune responses against disease-causing organisms or their associated toxins. These formulations are typically prepared with weakened forms of microbes, their surface proteins, or their virulence factors, which can train the immune system to recognize and neutralize similar infectious threats in later exposures. Owing to many unique properties of nanoparticles in enhancing vaccine potency, nanoscale carriers are drawing increasing interest as a platform for developing safer and more effective vaccine formulations. Notably, a nanoparticle-based strategy was recently demonstrated to safely deliver intact, non-denatured protein toxins to mount a potent anti-toxin immune response. A biomimetic nanoparticle cloaked in biological membranes was used to sequester membrane-active toxins. Upon interaction with the nanoparticles, the toxins become retrained and lose their toxicity as they are precluded from interacting with cellular targets. The resulting particle/toxin complex adopts a nanoparticulate morphology that facilitates the toxins' intracellular delivery. This sequestration approach has immense immunological implications owing to its ability in enabling structurally preserved toxins for immune processing. This technique offers opportunities in novel toxoid vaccine designs that promise more effective anti-toxin immune responses and contrasts the existing paradigm in toxoid preparation, in which toxins are antigenically altered to ensure virulence removal. The potent nanotoxoid formulations provide a viable anti-virulence measure in combating microbial infections that involve membrane-damaging toxins, including methicillin-resistant Staphylococcus aureus (MRSA) and Group A streptococcal infections.

The effect of organic acids on gelation characteristics of protein gels made from silver carp (Hypophthalmichthys molitrix) protein recovered by isoelectric solubilization and precipitation

Isoelectric solubilization and precipitation (ISP), a protein recovery process, was applied to ground, headed and gutted silver carp (Hypophthalmichthys molitrix) at both acidic and basic solubilization pH values using either glacial acetic acid (AA) or a combination of formic and lactic acid (F&L). Functional and textural properties of protein pastes and cooked gels made from recovered protein with standard additives were examined. Thermal denaturation and viscoelasticity of protein pastes were measured; pastes presented with endothermic reactions typical of non-denatured protein. Protein solubilized using AA and F&L resulted in endothermic properties similar to Alaska Pollock surimi. ISP-recovered protein presented with rheological characteristics typical of myofibrillar proteins from other marine protein sources. Viscoelastic properties of proteins solubilized at basic conditions using F&L had a high elastic modulus (G′), which is an indication of good gelation and gel quality. Shear stress on cooked gels was highest (p < 0.05) in gels made from protein solubilized at pH 11.5 using AA. Protein recovered by ISP processing retained their functionality and gels made from the protein were similar to surimi; therefore, potential exists for its use as a high quality protein source.

Synthesis of a Bridging Ligand with a Non-denatured Protein Pendant: Toward Protein Encapsulation in a Coordination Cage

Abstract Toward protein encapsulation by a synthetic host, we synthesized an ubiquitin-dangled ligand, a potential precursor of a nanoscale coordination cage. The key step is the addition of a C-terminal Cys76 SH group, which was introduced by Gly76Cys mutation, to a maleimide acceptor on the ligand. The C-terminal mutation and the SH addition to the ligand did not damage the ubiquitin moiety at all, neither structurally nor conformationally.

Properties of β-thioglucoside hydrolases (TGG1 and TGG2) from leaves of Arabidopsis thaliana

Myrosinases (β-thioglucoside glucohydrolase, TGG; EC 3.2.1.147) catalyze the hydrolysis of glucosinolates, a structurally distinct group of nitrogen- and sulfur-containing secondary metabolites, to give a chemically unstable intermediate, glucose and sulfate. This catalysis initiates a chemical defense in crucifer plants as a response to the tissue-damaging activities of herbivores and pathogens. To characterize the individual and collective biochemical properties of the myrosinase enzymes found in the aerial tissues of Arabidopsis thaliana, we purified TGG1 and TGG2, which share 73% amino acid identity, individually from T-DNA insertion lines of Arabidopsis using lectin affinity and anion exchange chromatography. Electrophoresis under denaturing conditions and the mobility of nondenatured TGG1 and TGG2 protein on gel filtration chromatography indicated that the native proteins exist as dimers of 150 and 126kDa, respectively.Despite their relatively similar kinetic parameters, both enzymes had distinct physicochemical properties such as extractability in low ionic strength buffer and electrophoretic mobility following deglycosylation treatment. Deglycosylation under nondenaturing conditions had limited effects on TGG1 and no effect on TGG2 activity. Both enzymes functioned across a broad range of temperatures (up to 60°C) and pH values (5–10). These results demonstrate that myrosinases have the ability to function in environments like the digestive tract of insect herbivores that are significantly different from the environment in a damaged plant.

Characterization of low viscosity polymer solutions for microchip electrophoresis of non-denatured proteins on plastic chips.

In this paper, we study characteristics of polymers (methylcellulose, hypromellose ((hydroxypropyl)methyl cellulose), poly(vinylpyrrolidone), and poly(vinyl alcohol)) with different chemical structures for microchip electrophoresis of non-denatured protein samples in a plastic microchip made of poly(methyl methacrylate) (PMMA). Coating efficiency of these polymers for controlling protein adsorption onto the channel surface of the plastic microchip, wettability of the PMMA surface, and electroosmotic flow in the PMMA microchannels in the presence of these polymers were compared. Also relative electrophoretic mobility of protein samples in solutions of these polymers was studied. We showed that when using low polymer concentrations (lower than the polymer entanglement point) where the sieving effect is substantially negligible, the interaction of the samples with the polymer affected the electrophoretic mobility of the samples. This effect can be used for achieving better resolution in microchip electrophoresis of protein samples.

Determination of gas phase protein ion densities via ion mobility analysis with charge reduction

We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1 : 49.5 : 49.5, formic acid : methanol : water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase measurements to be correlated to biomolecular structures in solution, low charge state ions should be analyzed. Further, to determine if different solution conditions give rise to ions of different structure, ions of similar charge state should be compared. Non-denatured protein ion densities are found to be in excellent agreement with non-denatured protein ion densities inferred from prior DMA and drift tube measurements made without charge reduction (all ions with densities in the 0.85-1.10 g cm(-3) range), showing that these ions are not strongly influenced by Coulombic stretching nor by analysis method.

Purification and characterization of mammalian glucose transporters expressed in Pichia pastoris

The major bottleneck to the application of high-resolution techniques such as crystallographic X-ray diffraction and spectroscopic analyses to resolve the structure of mammalian membrane proteins has been the ectopic expression and purification of sufficient quantities of non-denatured proteins. This has been especially problematic for members of the major facilitator superfamily, which includes the family of mammalian glucose transporters. A simple and rapid method is described for the purification of milligram quantities of recombinant GLUT1 and GLUT4, two of the most intensively studied GLUT isoforms, after ectopic expression in Pichia pastoris. The proteins obtained were >95% pure and exhibited functional transport and ligand-binding activities.

Semi-crosslinked polyacrylamides as high-resolution and dynamic self-coating sieving matrices for protein capillary electrophoresis

This paper describes non-gel capillary sieving electrophoresis employing semi-crosslinked polyacrylamide as a high performance and low viscous replaceable separation matrix for separation of non-denatured protein separation. Arising from the fine sieving and dynamic coating ability of this polymer, a mixture of basic proteins lysozyme, cytochrome C, ribonuclease A, and trypsin was resolved with excellent reproducibility. Mixing different semi-crosslinked polyacrylamides together further improves the separation. The separtion mechanism was analyzed. With network structure developed to an intermediate state between crosslinked gel and linear polymer solutions, these semi-crosslinked olyacrylamide polymers demonstrate a promise as a new class of size sieving separation medium, not only in capillary electrophoresis, but also in microfluidic chip separation schemes.

Identification of an N-Linked Glycosylation in the C4 Region of HIV-1 Envelope gp120 That Is Critical for Recognition of Neighboring CD4 T Cell Epitopes

The heavy glycosylation of HIV-1 envelope gp120 shields this important Ag from recognition by neutralizing Abs and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of gp120 epitopes and their recognition by MHC class II-restricted CD4 T cells. In this study, three conserved glycans (linked to N406, N448, and N463) flanking the C4 region of gp120 that contains many known CD4 T cell epitopes were disrupted individually or in combination by asparagine-to-glutamine substitutions. The mutant proteins lacking the N448 glycan did not effectively stimulate CD4 T cells specific for the nearby C4 epitopes, although the same mutants were recognized well by CD4 T cells specific for epitopes located in the distant C1 and C2 regions. The loss of recognition was not due to amino acid substitutions introduced to the mutant proteins. Data from trypsin digestion and mass spectrometry analyses demonstrated that the N448 glycan removal impeded the proteolytic cleavage of the nearby C4 region, without affecting more distant sites. Importantly, this inhibitory effect was observed only in the digestion of the native nondenatured protein and not in that of the denatured protein. These data indicate that the loss of the N448 glycan induces structural changes in the C4 region of gp120 that make this specific region more resistant to proteolytic processing, thereby restricting the generation of CD4 T cell epitopes from this region. Hence, N-linked glycans are critical determinants that can profoundly influence CD4 T cell recognition of HIV-1 gp120.

Effect of foaming temperature and varying time/temperature-conditions of pre-heating on the foaming properties of skimmed milk

The objective of this study was to investigate the effect of foaming temperature and impact of heat on the foaming properties of skimmed milk. Increasing foaming temperatures from 4 to 60 °C led to a decreased average foam bubble diameter ( d 10) from 0.26 to 0.18 mm. Temperature increase also led to increased foam density values (0.14–0.22 g cm −3). Overall, the skimmed milk foam that was prepared at 50–60 °C was the most stable, while minimal drainage was detected in the range of 30–40 °C. Pre-heating of skimmed milk was performed by high-temperature-short-time (HTST), high temperature (HT)- or ultra-high-temperature (UHT)-heating. The ( d 10)-values of the milk foam increased from non-heated to HT-heated milk. Foam density decreased in a continuous fashion from unheated to UHT-heated milk. The composition of non-denatured whey proteins in milk samples and the respective foams differed only marginally. Foam from unheated milk was the most stable. Transmission electron microscope-images revealed differences in foam microstructure in relation to the age of the foam and the heat treatment applied. The air–serum interface in the foam of skimmed milk consists of casein and whey protein monomers to oligomers. Casein micelles do not seem to be an integral part of the air–serum interface in skimmed milk foam.

Dynamic Cross-Linking Effect of Mg2+ To Enhance Sieving Properties of Low-Viscosity Poly(vinylpyrrolidone) Solutions for Microchip Electrophoresis of Proteins

We report a dynamic cross-linking effect of Mg2+ that enhances the sieving properties of low-viscosity poly(vinylpyrrolidone) (PVP) solutions. A low-viscosity PVP solution was applied to nondenaturing microchip electrophoresis of protein samples using microchips made of poly(methyl methacrylate). The separation resolution of nondenatured protein markers in 1.8% PVP solution was improved by adding 1-20 mM MgCl2. We studied the effect of the ratio of cross-linking agent on mobility of protein samples and showed that protein retardation (ln micro/micro0) is correlated with the ratio of cross-linking agent to PVP ([cMg2+/cPVP]) as ln micro/micro0=A'[cMg2+/cPVP]b'. A' was related to the protein radius (R), and b' was found to be 0.72 for proteins with R=2.4 nm and 0.82 for proteins with R=1.85 nm. A structural study of PVP in semidilute solutions using dynamic light scattering showed that incremental increases of Mg2+ ion concentration from 5 to 20 mM in 1.8% PVP solution increased the hydrodynamic radius of PVP polymers by 20%.

The Medicago truncatula Small Protein Proteome and Peptidome

The small protein and native peptide component of plant tissues is a neglected area of proteomic studies. We have used fractionation techniques for denatured and nondenatured protein preparations combined with 2-D LC tandem mass spectrometry to examine the sequences of small proteins and peptides in four tissues of the model legume, Medicago truncatula: the root tip and root of germinating seedlings, nitrogen fixing nodules, and young leaves. The isolation and fractionation strategies successfully enriched the small protein and native peptide content of the samples. Eighty-one small M. truncatula proteins and native peptides were identified. Most samples were dominated by ribosomal and histone proteins, and leaf samples possessed photosynthesis-related proteins. Secreted proteins such as lipid transfer proteins were common to several tissues. Twenty-four hours after germination, the roots and root tip tissues possessed several "seed-specific" and late-embryogenesis proteins. We conclude that these proteins are present in cells prior to germination and that they are subsequently used as a nutritional source for the young tissues. Native UV absorbing peptides were detected in very low molecular weight fractions and sequenced. Each peptide shared C-terminal residues and showed homology to the seed storage protein legumin. The strategies used here would be suitable for combining bioassays and mass spectrometry to identify bioactive peptides in the M. truncatula peptidome.

Proteomic Identification of M. tuberculosis Protein Kinase Substrates: PknB Recruits GarA, a FHA Domain-containing Protein, Through Activation Loop-mediated Interactions

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.

XRF and XAFS analysis of electrophoretically isolated nondenatured proteins

Microbial metal reduction is relevant for developing bioremediation strategies for contaminated sites containing toxic metal compounds, but the molecular mechanisms involved and the regulation of those mechanisms are poorly understood. Metal reduction capability is microbe specific, yet the involvement of metalloproteins in the transport of electrons from electron donor molecules to electron acceptor molecules (i.e., to the metals to be reduced) is common to all metal-reducing organisms. A thorough understanding of the mechanisms involved in metal reduction by microbes, with a focus on their metalloproteins, will provide valuable information about the metal reduction capability of the organisms and could provide general insight into the mechanisms of metalloprotein expression and regulation in other microbial systems as well. We are developing methods for the efficient separation, detection, and quantification of metalloproteins. Electrophoretic methods for protein separation have been combined with x-ray fluorescence mapping and XAFS to determine the abundance and local chemical environment of iron within a cytochrome.

Prediction of the Charge States of Folded Proteins in Electrospray Ionization

Earlier work from this laboratory dealt with the observation that the charge states of non-denatured proteins can be decreased by use of buffer salts in which the gas-phase basicity of conjugate base B, GB(B), of the buffer cations is high. A theoretical model was developed and applied to several small proteins. The predictions of the charge states were found to be in good agreement with those observed experimentally. Because the computational model is based on the charge residue model (CRM), the observed agreement lends support for the CRM. In the present work, the same model is applied to recent data by Catalina et al. who showed that very large charge reductions are achieved with very high GB(B) proton sponges. Their data included lysozyme but also the very much larger proteins, p-hydroxybenzoate hydroxylase (PHBH), 90 kDa and glutamate synthase (GLTS), 166 kDA. The present work examines the performance of the model for the much stronger bases and the very much larger proteins. It is found that the predictions of the charge states agree well for the small protein lysozyme but somewhat less well with the experimental results for PHBH and GLTS. The causes for the lack of good agreement with the large proteins are examined.

Study of the strawberry Cel1 endo-β-(1,4)-glucanase protein accumulation and characterization of its in vitro activity by heterologous expression in Pichia pastoris

Strawberry ( Fragaria x ananassa Duch.) fruit ripening is associated with a marked loss of firm texture, and endo-β-(1,4)-glucanases (EGases; EC 3.2.1.4) are believed to contribute to this loss process. To study the role of the previously described EGase cel1 in strawberry fruit softening, polyclonal antibodies have been raised against its protein. Immunoblot analyses have demonstrated that Cel1 protein is ripening related and that its accumulation is repressed by the auxin analogue 1-naphtalene acetic acid (NAA). Isoforms of EGases in non-denatured protein extracts from strawberry fruit were fractionated by native IEF and visualised with specific activity staining. Using this approach, we have obtained evidence for the existence of five carboxymethylcellulase (CMCase) isoforms, which differed in their isoelectric point. Western-blot analysis revealed that one of these isoforms, with basic isoelectric point, corresponded to strawberry Cel1 protein. Over-expression of Cel1 in the yeast Pichia pastoris revealed that the recombinant protein has an optimum pH activity of 7.5 and acts against different β-(1,4) cell wall polymers, including carboxymethylcellulose (CMC) and cellulose.

An epitope tag derived from human transcription factor IIB that reacts with a polyol-responsive monoclonal antibody

Polyol-responsive monoclonal antibodies (PR-mAbs) provide a strategy to purify active, nondenatured proteins by a single-step immunoaffinity chromatography procedure. The high affinity interaction between these antibodies and the antigen can be dissociated in the presence of a nonchaotropic salt and a low molecular weight polyhydroxylated compound (polyol). The epitope for PR-mAb IIB8 is located near the N-terminus of the human transcription factor IIB (TFIIB). The epitope is an eight amino acid sequence, TKDPSRVG, that can be fused to a desired protein for use as a purification tag. This epitope tag (termed hIIB) was fused to the C-terminus of green fluorescent protein (GFP). An additional GFP fusion protein utilized another version of hIIB containing a point mutation at position two. These fusion proteins, expressed in Escherichia coli, allowed successful separation of the desired protein in a single chromatographic step. This strategy extends PR-mAb gentle-release purification to numerous expressed proteins.

Utilization of plastic tubes for the first dimension of two-dimensional gel electrophoresis

A new technique for tube-gel isoelectric focusing (IEF) using plastic tubes instead of glass tubes in the first dimension of two-dimensional gel electrophoresis (2-DE) was developed for proteomic analysis of both denatured and non-denatured proteins. The plastic tubes have advantages in handling gels over glass tubes. The plastic tubes were also applied successfully to native gel electrophoresis, blue-native gel electrophoresis, agarose gel IEF, acid-urea gel electrophoresis and cetyltrimethylammonium bromide (CTAB) gel electrophoresis for the first dimension of 2-DE.

Heat-induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics.

The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly pure ovalbumin, the decrease in nondenatured native protein showed first-order dependence. The activation energy obtained with different techniques varied between 430 and 490 kJ*mole(-1). First-order behavior was studied in detail using differential scanning calorimetry. The calorimetric traces were irreversible and highly scan rate-dependent. The shape of the thermograms as well as the scan rate dependence can be explained by assuming that the thermal denaturation takes place according to a simplified kinetic process where N is the native state, D is denatured (or another final state) and k a first-order kinetic constant that changes with temperature, according to the Arrhenius equation. A kinetic model for the temperature-induced denaturation and aggregation of ovalbumin is presented. Commercially obtained ovalbumin was found to contain an intermediate-stable fraction (IS) of about 20% that was unable to form aggregates. The denaturation of this fraction did not satisfy first-order kinetics.

Effect of buffer cations and of H3O+ on the charge states of native proteins. Significance to determinations of stability constants of protein complexes.

The progressive reduction of charge in charge states of non-denatured proteins (lysozyme, ubiquitin, and cytochrome c), observed with nanospray in the positive ion mode, when the buffer salt ammonium acetate is replaced by ethylammonium acetates (EtNH(3)Ac, Et(2)NH(2)Ac and Et(3)NHAc) is rationalized on the basis of the charge residue model (CRM). The charge states of the multiply protonated protein are shown to be controlled by the increasing gas-phase basicities, GB(B), of the bases(B) NH(3), EtNH(2), Et(2)NH and Et(3)N. Charge states derived from evaluated apparent gas-phase basicities GB(app) of the basic side-chains of the protein and the known GB(B) of the above bases are found to be in agreement with the experimentally observed charge states. This is a requirement of the CRM, because in this model the small positive ions (the buffer cations in the present case) at the surface of the electrospray droplets are the excess ions that provide the charge of the final small droplet that contains the protein molecule and on evaporation of the solvent transfer the charge to the protein. The observed charge states in the absence of buffer salts, i.e. pure water, are attributed to excess H(3)O(+) ions produced by the electrolysis process that attends electrospray. A proposed extended mechanism provides predictions of factors that determine the sensitivity for detection of the multiply protonated proteins. Consideration of restraints imposed by the CRM lead to some simple predictions for conditions that should be present to obtain accurate determinations by electrospray and nanospray of stability constants for the protein-complex equilibrium in aqueous solution.