Anionic graft polymerization of methyl acrylate to protein functional groups

Graft polymerization of methyl acrylate to functional groups in proteins was studied with model compounds and with whole gluten proteins. Polymerization was carried out in the presence of sodium hydride or sodium in dimethyl sulfoxide. Initiation proceeds by an anionic mechanism, and the rate-determining step is the production of the initially formed carbanion. The rate of disappearance of methyl acrylate was followed via gas chromatography. Amino acid analyses indicated that the functional groups of the amino acids, as well as the peptide bonds, were acting as the initiation sites in proteins. Reaction rates of the functional groups were determined on model compounds in the presence of sodium and sodium hydride. With both the model compounds and the proteins, polymerization was initially rapid and then leveled off, although rates depended on the concentration of activator and acrylate. Methoxyl group analyses of modified model compounds and proteins indicated that from 5 to 10 methyl acrylate residues were introduced per reactive site.     

Liquid chromatography with complementary electrospray and inductively coupled plasma mass spectrometric detection of ferrocene-labelled peptides and proteins

Succinimidylferrocenyl propionate (SFP) is introduced as labelling agent for amino functions in peptides and proteins. The resulting derivatives are characterised by considerably lower polarity compared with the native analytes and can thus be well separated by means of reversed phase liquid chromatography (RP-LC). The reaction products are characterised by electrospray ionisation mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). A further advantage of the method is a simple and straightforward derivatisation protocol. Different basic and acidic model proteins as lysozyme, ß-lactoglobulin A and insulin were derivatised using SFP. Furthermore, the first dual-labelling strategy of thiol and amino groups with ferrocene-based reagents is presented. Whereas the amino groups were derivatised with SFP, the thiol groups were functionalised by reaction with ferrocenecarboxylic acid(2-maleimidoyl)ethylamide. Again, LC/ESI-MS is a suitable tool to characterise the modified peptides and proteins.     

Site directed maleimide bifunctional chelators for the M(CO)3+ core (M =(99m)Tc, Re)

A series of bifunctional chelates containing a tridentate donor set for complexation of the M(CO)3+ core and a maleimide group for site-specific coupling to peptides and proteins containing free thiol groups has been prepared and their Re(CO)3+ complexes and glutathione conjugates structurally characterized. The flexibility of design allows preparation of ligands suitable for both fluorescence imaging, radioimaging and radiotherapeutic studies of proteins and peptides as well as other biopolymers using site specific conjugation.     

High-performance liquid chromatography of proteins using chemically-modified silica supports

Summary Highly efficient and fast exclusion-chromatographic separations of proteins are possible on chemically-modified, silica stationary phases. By optimizing the pH and the ionic strength of the aqueous eluent secondary interactions of the samples with surface groups can be excluded. Bonded propylamide groups proved to possess optimum properties for exclusion chromatography. With other functional groups adsorption effects cannot be excluded totally. The optimum pore size distribution for protein separation up to relative molecular masses of 500,000 daltnons is between 10nm and 50nm. With these silica-based phases the pore size distribution, the pore volume and the packing characteristics are independent of the eluent, therefore the same column can be used with aqueous as well with organic eluents. It is possible to correlate the elution volume (molecular size) of proteins with those of polystyrene standars. The recovery of the proteins and their biological activity has always been better than 90%. The potentialities of adsorption chromatography of proteins on chemically-bonded stationary plases with different functional groups are demonstrated.     

Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties

Recent efforts at the proteomic level were employed to describe the protein equipment of the plasma membrane of the model plant Arabidopsis thaliana. These studies had revealed that the plasma membrane is rich in extrinsic proteins but came up against two major problems: (i) few hydrophobic proteins were recovered in two-dimensional electrophoresis gels, and (ii) many plasma membrane proteins had no known function or were unknown in the database despite extensive sequencing of the Arabidopsis genome. In this paper, several methods expected to enrich a membrane sample in hydrophobic proteins were compared. The optimization of solubilization procedures revealed that the detergent to be used depends on the lipid content of the sample. The corresponding proteomes were compared with the statistical model AMMI (additive main effects with multiplicative interaction) that aimed at regrouping proteins according to their solubility and electrophoretic properties. Distinct groups emerged from this analysis and the identification of proteins in each group allowed us to assign specific features to several of them. For instance, two of these groups regrouped very hydrophobic proteins, one group contained V-ATPase subunits, another group contained proteins with one transmembrane domain as well as proteins known to interact with membrane proteins. This study provides methodological tools to study particular classes of plasma membrane proteins and should be applicable to other cellular membranes.     

IMAC—Immobilized metal ion affinity based chromatography

Immobilized metal ion affinity chromatography (IMAC) is a highly versatile separation method based on interfacial interactions between biopolymers in solution and metal ions fixed to a solid support, which is usually a hydrophilic cross-linked polymer.Polymer-fixed Zn(II), Ni(II), Co(II) and Cu(II) are particularly well suited for fractionation of proteins primarily on the basis of their relative content of surface-located imidazole residues but also of Trp and Cys residues as well as terminal amino groups.IMA methods can also be devised for purification of phosphoproteins and calcium-binding proteins. In some instances, the performance of IMA gels is comparable to that of biospecific affinity-based adsorbents. In fact IMA gels may, by sandwich techniques, occasionally be converted to biospecific adsorbents.Selectivity can be varied by choice of type of ligand and metal ion as well as by varying the modes of elution, including affinity desorption.     

Adhesion forces between functionalized latex microspheres and protein-coated surfaces evaluated using colloid probe atomic force microscopy

Proteins are important in bacterial adhesion, but interactions at molecular-scales between proteins and specific functional groups are not well understood. The adhesion forces between four proteins [bovine serum albumin (BSA), protein A, lysozyme, and poly-d-lysine] and COOH, NH2 and OH-functionalized (latex) colloids were examined using colloid probe atomic force microscopy (AFM) as the function of colloid residence time (T) and solution ionic strength (IS). For three of the proteins, OH-functionalized colloids produced higher adhesion forces to proteins (2.6-30.5 nN; IS=1 mM, T=10s) than COOH- and NH2-functionalized colloids (1.6-6.8 nN). However, protein A produced the largest adhesion force (8.1+/-1.0 nN, T=10 s) with the COOH-functionalized colloid, demonstrating the importance of specific and unanticipated protein-functional group interactions. The NH2-functionalized colloid typically produced the lowest adhesion forces with all proteins, likely due to repulsive electrostatic forces and weak bonds for NH2-NH2 interactions. The adhesion force (F) between functionalized colloids and proteins consistently increased with residence time (T), and data was well fitted by F=ATn. The constant value of n=0.21+/-0.07 for all combinations of proteins and functionalized colloids indicated that water exclusion and protein rearrangement were the primary factors affecting adhesion over time. Adhesion forces decreased inversely with IS for all functional groups interacting with surface proteins, consistent with previous findings. These results demonstrate the importance of specific molecular-scale interactions between functional groups and proteins that will help us to better understand factors colloidal adhesion to surfaces.     

Perfluoroaryl and Perfluoroheteroaryl Reagents as Emerging New Tools for Peptide Synthesis,Modification and Bioconjugation

Peptides and proteins are becoming increasingly valuable as medicines, diagnostic agents and as tools for biomedical sciences. Much of this has been underpinned by the emergence of new methods for the manipulation and augmentation of native biomolecules. Perfluoroaromatic reagents are perhaps one of the most diverse and exciting tools with which to modify peptides and proteins, due principally to their nucleophilic substitution chemistry, high electron deficiency and the ability for their reactivity to be tuned towards specific nucleophiles. As discussed in this minireview, in recent years, perfluoroaromatic reagents have found applications as protecting groups or activating groups in peptide synthesis and as orthogonal handles for peptide modification. Furthermore, they have applications in chemoselective ‘tagging’, stapling and bioconjugation of peptides and proteins, as well as tuning of ‘drug-like’ properties. This review will also explore possible future applications of these reagents in biological chemistry.     

维拉帕米作用下急性心梗大鼠比较蛋白质组学研究

以急性心梗大鼠为研究对象, 应用双向凝胶电泳法(2-DE)分析比较了维拉帕米作用下急性心梗大鼠心肌蛋白表达的差异, 从蛋白质水平探讨了维拉帕米心肌保护作用的发生机制. 结果表明, 与假手术组及模型组相比, 维拉帕米给药组心肌组织中有8个蛋白点表达显著上调, 7个蛋白点表达显著下调. 采用质谱(MALDI-TOF-MS)分析结合数据库检索, 共鉴定了其中的15种蛋白质, 可按功能分为如下4类: (1) 能量代谢及线粒体功能相关蛋白; (2) 氧化应激相关蛋白; (3) 细胞骨架蛋白; (4) 其它蛋白. 研究结果表明, 维拉帕米的心肌保护作用与恢复心肌损伤过程中的能量供应及对抗氧化应激等作用有关.     

The role of ligands on protein retention in adsorption chromatography: A surface energetics approach

Protein adsorption onto hydrophobic chromatographic supports has been investigated using a colloid theory surface energetics approach. The surface properties of commercially available chromatographic beads, Toyopearl Phenyl 650‐C, and Toyopearl Butyl 650‐C, have been experimentally determined by contact angle and zeta potential measurements. The adsorption characteristics of these beads, which bear the same backbone matrix but harbor different ligands, have been studied toward selected model proteins, in the hydrated as well as dehydrated state. There were two prominent groups of proteins observed with respect to the chromatographic supports presented in this work: loosely retained proteins, which were expected to have lower average interaction energies, and the strongly retained proteins, which were expected to have higher average interaction energies. Results were also compared and contrasted with calculations derived from adsorbent surface energies determined by inverse liquid chromatography. These results showed a good qualitative agreement, and the interaction energy minima obtained from these extended Derjaguin, Landau, Verwey and Overbeek calculations were shown to correlate well with the experimentally determined adsorption behavior of each protein.     

Branched polyethyleneimine-bonded tentacle-type polymer stationary phase for peptides and proteins separations by open-tubular capillary electrochromatography

A novel tentacle-type polymer stationary phase covalently modified with branched polyethyleneimine (PEI) was developed for peptides and proteins separations by open-tubular CEC (OT-CEC). The preparation procedure included the silanization of capillary inner wall, in situ graft polymerization and PEI functionalization. A wrinkly polymer surface of multitudinous steric amine groups was evenly formed on the capillary inner wall, and anodic EOF could be gained within a wide pH range of 2.5-7.5. The electroosmotic mobility was examined for its dependence on pH as well as PEI concentrations. Good repeatability was gained with RSD for the migration time of EOF marker within 4.8% and satisfactory chemical stability was validated. Due to the existence of amine groups on the surface of tentacle-type polymer stationary phase, the silanol effect that occurs between the positively charged biomolecules and the silanols of the capillary column was greatly suppressed. Compared with a monolayer-coating capillary, seven enkephalin-related peptides were well resolved on the PEI-bonded column with high efficiencies. Favorable separations of peptides and proteins with high column efficiencies were obtained in 144,000-189,000 and 97,000-170,000 plates/m. Branched PEI-bonded tentacle-type polymer stationary phase has been proven to afford satisfactory retention and resolution of peptides and proteins.     

Retention of proteins and metalloproteins in open tubular capillary electrochromatography with etched chemically modified columns

Etched chemically modified capillaries with two different bonded groups (pentyl and octadecyl) are compared for their migration behavior of several common proteins and metalloproteins as well as metalloproteinases. Migration times, efficiency and peak shape are evaluated over the pH range of 2.1-8.1 to determine any effects of the bonded group on the electrochromatographic behavior of these compounds. One goal was to determine if the relative hydrophobicity of the stationary phase has a significant effect on proteins in the open tubular format of capillary electrochromatography as it does in HPLC. Reproducibility of the migration times is also investigated.     

Homocysteine thiolactone and protein homocysteinylation: mechanistic studies with model peptides and proteins

In vitro incubations were performed to show that homocysteine thiolactone could generate covalent adducts with model peptides and proteins. MS and MS/MS data suggest that the thiolactone reacts with the side-chain amino group of lysine residues as well as with the N-terminal amino group or C-terminal carboxy group. For larger peptides and proteins, the contribution from the in-amino groups of lysine residues should be predominant. These data could help explain the detrimental effects of elevated levels of homocysteine and homocysteine thiolactone.     

Benchmarking pKaprediction

Background  

pK_avalues are a measure of the protonation of ionizable groups in proteins. Ionizable groups are involved in intra-protein, protein-solvent and protein-ligand interactions as well as solubility, protein folding and catalytic activity. The pK_ashift of a group from its intrinsic value is determined by the perturbation of the residue by the environment and can be calculated from three-dimensional structural data.     

Defining the plant disulfide proteome

There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.     

Biomimetic seleninates and selenonates

The synthesis of a variety of pyranose-, nucleoside-, (amino acid)-, and polyhydric-based seleninic and selenonic acids by DMDO oxidation of the corresponding selenoesters is reported, as well as some unusual coupling reactions of the seleninate and selenonate functionality with biological nucleophilic groups (sulfhydryl, indole, phenol, imidazole, carboxamide) that are found in proteins and enzyme active sites.     

Dual mode bioreactions on polymer nanoparticles covered with phosphorylcholine group

We investigated the preparation of polymer nanoparticles covered with phosphorylcholine (PC) groups and the immobilization of proteins in order to observe dual mode bioreactions on the nanoparticles. For the surface modification on the nanoparticles, a water-soluble amphiphilic phospholipid polymer with PC groups as a hydrophilic moiety was synthesized. In this polymer, an active ester group, which can immobilize proteins, was introduced. Using the phospholipid polymer as a solubilizer, poly(L-lactic acid) nanoparticles were prepared from its methylene chloride solution in an aqueous medium by the solvent evaporation method. The diameter of the nanoparticles was ca. 200 nm and the surface was covered with the PC groups and active ester groups. Proteins could immobilize on the nanoparticles under mild conditions by the reaction between the active ester group and amino group in the proteins. Both an antibody and enzyme were immobilized on the nanoparticles and bioreactions such as the antigen/antibody reaction and enzymatic reaction were observed. When an antigen was added to the suspension of the nanoparticles, aggregation of the nanoparticles occurred and then they precipitated. Also, the enzymatic reaction proceeded well when the enzyme substrate was added to the suspension. Based on these results, we provided polymer nanoparticles functionalized with both the antibody and enzyme, and the dual mode bioreactions could occur. We concluded that the novel polymer nanoparticles could be used for nano-/micro-scaled diagnostic and medical treatment systems.     

Influence of surface modification on protein retention in ion-exchange chromatography: Evaluation using different retention models

A large number of different stationary phases for ion-exchange chromatography (IEC) from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (α-lactalbumin, β-lactoglobulin A, bovine serum albumin and alcohol dehydrogenase) were determined for three anion-exchange adsorbents based on synthetic copolymer beads with differences in the functional group chemistry. Fractogel EMD DEAE and Fractoprep DEAE consist of functional groups bound to the surface via “tentacles”, ToyopearlDEAE by a short linker. Three models which describe chromatographic retention were used to analyse the characteristic parameters of the protein/stationary-phase interactions. The number of electrostatic interaction between the stationary phase and the model proteins, the protein specific surface charge densities and the interacting surface of the proteins with the adsorptive layer of the chromatographic media depend on the surface modification as well as on the molecular mass of the model proteins. In general, protein retention of the model proteins on the weak anion exchangers was found to be greater if the stationary phase carries tentacles and protein mass is above 60 kDa.     

A Cysteine-Directed Proximity-Driven Crosslinking Method for Native Peptide Bicyclization

Efficient and site-specific modification of native peptides and proteins is desirable for synthesizing antibody-drug conjugates as well as for constructing chemically modified peptide libraries using genetically encoded platforms such as phage display. In particular, there is much interest in efficient multicyclization of native peptides due to the appeals of multicyclic peptides as therapeutics. However, conventional approaches for multicyclic peptide synthesis require orthogonal protecting groups or non-proteinogenic clickable handles. Herein, we report a cysteine-directed proximity-driven strategy for the constructing bicyclic peptides from simple natural peptide precursors. This linear to bicycle transformation initiates with rapid cysteine labeling, which then triggers proximity-driven amine-selective cyclization. This bicyclization proceeds rapidly under physiologic conditions, yielding bicyclic peptides with a Cys-Lys-Cys, Lys-Cys-Lys or N-terminus-Cys-Cys stapling pattern. We demonstrate the utility and power of this strategy by constructing bicyclic peptides fused to proteins as well as to the M13 phage, paving the way to phage display of novel bicyclic peptide libraries.     

Multitargeting by curcumin as revealed by molecular interaction studies

Curcumin (diferuloylmethane), the active ingredient in turmeric (Curcuma longa), is a highly pleiotropic molecule with anti-inflammatory, anti-oxidant, chemopreventive, chemosensitization, and radiosensitization activities. The pleiotropic activities attributed to curcumin come from its complex molecular structure and chemistry, as well as its ability to influence multiple signaling molecules. Curcumin has been shown to bind by multiple forces directly to numerous signaling molecules, such as inflammatory molecules, cell survival proteins, protein kinases, protein reductases, histone acetyltransferase, histone deacetylase, glyoxalase I, xanthine oxidase, proteasome, HIV1 integrase, HIV1 protease, sarco (endo) plasmic reticulum Ca(2+) ATPase, DNA methyltransferases 1, FtsZ protofilaments, carrier proteins, and metal ions. Curcumin can also bind directly to DNA and RNA. Owing to its β-diketone moiety, curcumin undergoes keto-enol tautomerism that has been reported as a favorable state for direct binding. The functional groups on curcumin found suitable for interaction with other macromolecules include the α, β-unsaturated β-diketone moiety, carbonyl and enolic groups of the β-diketone moiety, methoxy and phenolic hydroxyl groups, and the phenyl rings. Various biophysical tools have been used to monitor direct interaction of curcumin with other proteins, including absorption, fluorescence, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy, surface plasmon resonance, competitive ligand binding, Forster type fluorescence resonance energy transfer (FRET), radiolabeling, site-directed mutagenesis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), immunoprecipitation, phage display biopanning, electron microscopy, 1-anilino-8-naphthalene-sulfonate (ANS) displacement, and co-localization. Molecular docking, the most commonly employed computational tool for calculating binding affinities and predicting binding sites, has also been used to further characterize curcumin's binding sites. Furthermore, the ability of curcumin to bind directly to carrier proteins improves its solubility and bioavailability. In this review, we focus on how curcumin directly targets signaling molecules, as well as the different forces that bind the curcumin-protein complex and how this interaction affects the biological properties of proteins. We will also discuss various analogues of curcumin designed to bind selective targets with increased affinity.