The reaction of protein amino groups with methyl 5-iodopyridine-2-carboximidate. A possible general method of preparing isomorphous heavy-atom derivatives of proteins

1. The synthesis of methyl 5-iodopyridine-2-carboximidate and its reaction with amino groups of model compounds and performic acid-oxidized insulin are described. The reagent was designed to introduce heavy atoms into specific sites in proteins. 2. Specific reaction with the amino groups of oxidized insulin can be achieved under reasonably mild conditions giving rise to the corresponding N-monosubstituted amidines. 3. The extent of reaction of this reagent with protein amino groups can be readily determined by difference spectroscopy. Modification of lysine residues inhibits tryptic cleavage at such residues, and this can be of assistance in establishing the site of modification in the primary structure. 4. Evidence is presented to show that methyl 5-iodopyridine-2-carboximidate can react specifically, at pH5.0, with the aromatic amino group of 3-amino-l-tyrosine; the final product of this reaction is a 2-arylbenzoxazole. 5. The use of this reagent as a general method for preparing heavy-atom isomorphous derivatives of proteins is discussed.     

Selective labeling of selenomethionine residues in proteins with a fluorescent derivative of benzyl bromide

Benzyl bromide is used as a reagent for the selective modification of methionine residues in proteins. We here explored the suitability of the bromobenzyl moiety as a reactive group for the targeted fluorescent labeling of methionine and selenomethionine residues in proteins. A novel labeling reagent (N,N',N'-trimethyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- N'-(p-bromomethylbenzyl)-ethylenediamine, NBD-BBr) was synthesized and tested for reactivity with two model proteins containing single methionine or selenomethionine residues. The amounts of reagent and reactions times required for modification of methionine resulted in side reactions with other amino acid residues, a finding which was also confirmed for benzyl bromide itself. However, with selenomethionine, lower concentrations and shorter reaction times were sufficient for NBD-BBr modification. Under these conditions, labeling was confined to selenomethionine residues with one but not the other model protein. Where applicable, the protein labeling strategy characterized here is rapid and efficient. It should be useful in combination with cysteine-specific labeling if dual site-specific modification is desired.     

Dual-targeted labeling of proteins using cysteine and selenomethionine residues

A new strategy for dual site-selective labeling of proteins that uses metabolically incorporated selenomethionine as a target for covalent modification by iodoacetamide derivatives, forming selenonium salts, is described. In the absence of free cysteine, labeling is specific and efficient. Dual-targeted labeling of a protein can be achieved with combinations of unique cysteine and methionine residues, if the cysteine is labeled first with a maleimide or another reagent that does not react with the selenomethionine. The method should be useful in biophysical applications such as fluorescence energy transfer.     

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.     

Immunochemical detection of adenine nucleotide-binding proteins with antibodies to 5'-p-fluorosulfonylbenzoyladenosine

5'-p-Fluorosulfonylbenzoyladenosine (FSBA) is a useful reagent for the affinity labeling of adenine nucleotide binding proteins. We have developed an immunochemical approach to the detection of proteins that have been covalently modified with FSBA, which provides an alternative to the use of a radiolabeled ligand. Antibodies have been prepared against FSBA-modified glutamate dehydrogenase and purified by chromatography on ATP-agarose. The resulting affinity-purified antibodies react on Western blots only with proteins that have been labeled previously with the affinity reagent. The degree of immunoreactivity on Western blots correlates well with the extent of covalent modification as shown by studies on the modification and inhibition of the catalytic subunit of cAMP-dependent protein kinase. In crude cellular extracts, numerous proteins can be labeled with FSBA and then detected by using this approach. The labeling and subsequent detection of these proteins can be blocked by including an excess of MgATP, which competes with FSBA for nucleotide-binding sites. The labeling of specific proteins in crude mixtures is saturable, as shown by labeling studies of p56lck, a protein-tyrosine kinase that is abundantly expressed in membranes from the T lymphoma cell line LSTRA.     

Affinity modification in a proteomic study of DNA repair ensembles

The review concerns the use of the affinity modification method as an integral part of the modern proteomic analysis to search for and identification of proteins belonging to protein ensembles of DNA repair. Affinity modification is based on the preliminary formation of specific non-covalent complex between the target biopolymer and a reagent (chemically reactive analog of biopolymer or low molecular weight ligand) followed by formation of covalent bond between the reagent and the site of the target, to which the reagent is bound, that ensures the method specificity. This method is most widely and effectively used in the study of structural and functional aspects of protein-nucleic acids interactions. Upon construction of DNA probes, in addition to chemically reactive groups and structural elements involved in specific recognition of DNA by proteins, additional groups that facilitate the subsequent affinity isolation of DNA-protein cross-links, can be introduced into the reagent. The review covers recent examples affinity DNA-reactive probe in combination with mass spectrometric and immunological methods to search for and identification in cell extracts, proteins interacting with apurinic/apyrimidinic sites and the proteins recognizing the cross-links in DNA induced by cisplatin.     

Current status of protein chip development in terms of fabrication and application

Sequencing of the human genome revealed that more than 30 000 genes encode proteins comprising the human proteome. "Proteomics" can be defined as a field of research studying proteins in terms of their function, expression, structure, modification and their interaction in physiological and in pathological states. The concentration, modification and interaction of proteins in cells, plasma, and in tissues are crucial in determining the phenotype of living organisms. Although fluctuation of protein concentration is essential to maintain homeostasis, protein expression levels are also pathognomonic features. Estimating protein concentration by analyzing the quantity of mRNA in cells through conventional technologies, such as DNA chips, does not provide precise values since the half-life and translation efficacy of mRNA is variable. In addition, polypeptides undergo post-translational modification. For these reasons, novel techniques are needed to analyze multiple proteins simultaneously using protein microarrays. In the near future, protein chips may allow construction of complete relational databases for metabolic and signal transduction pathways. This article reviews the current status of technologies for fabricating protein microarrays and their applications.     

白细胞介素－2的PEG化

用自制的氨基ＰＥＧ化试剂ｒＩＬ－２进行化学修饰,研究了试剂浓度,溶液ｐＨ,反应时间等与ＰＥｃａ－ｒＩＬ－２产率及ＩＬ－２活性保持之间的关系,建立了一套获得稳定修饰度的ＰＥＧ－ｒＩＬ－２的方法。研究发现,反应时间跟修饰度关系不大;溶液ｐＨ对修饰度有一定的影响,中性ｐＨ以上反应都可进行;而试剂浓度直接决定修饰度的高低,过量越多,修饰度越高,而生物活性保留也越低;但低度修饰,对活性几乎没有影响,可保留活性在９５％左右。     

Analysis of tryptophan surface accessibility in proteins by MALDI-TOF mass spectrometry

Surface accessible amino acids can play an important role in proteins. They can participate in enzyme's active center structure or in specific intermolecular interactions. Thus, the information about selected amino acids' surface accessibility can contribute to the understanding of protein structure and function. In this paper, we present a simple method for surface accessibility mapping of tryptophan side chains by their chemical modification and identification by MALDI-TOF mass spectrometry. The reaction with 2-hydroxy-5-nitrobenzyl bromide, a common and highly specific covalent modification of tryptophan, seems to be very useful for this purpose. The method was tested on four model proteins with known spatial structure. In the native proteins (1) only surface accessible tryptophan side chains were found to react with the modification agent and (2) no buried one was found to react at lower reagent concentrations. These results indicate that the described method can be a potent tool for identification of surface-located tryptophan side chain in a protein of unknown conformation.     

Preparation of protein conjugates via intermolecular disulfide bond formation

Conjugates of two unlike proteins can be prepared via the intermolecular disulfide interchange reaction, namely, protein A containing thiol groups reacts with protein B containing 4-dithiopyridyl groups to yield a conjugate with the release of 4-thiopyridone. Thiol groups can be introduced into proteins upon amidination with methyl 3-mercaptopropionimidate ester or 2-iminothiolane, and 4-dithiopyridyl groups can be introduced into proteins with these same reagents in the presence of 4,4'-dithiodipyridine. 2-Iminothiolane is stable on storage in contrast to the known lability of imidate esters; therefore 2-iminothiolane is a more convenient reagent for the modification of protein than are the imidate esters. All the reactions can be carried out easily under mild conditions in good yields. Conjugates of bovine plasma albumin with itself, ribonuclease, or a copolymer of D-glutamic acid and D-lysine and of sheep antibody and horseradish peroxidase were prepared with modified proteins containing an average of 1 to 5 thiol or dithiopyridyl groups per mol. These conjugates formed mainly dimers, trimers, and tetramers. The peroxidase labeled antibody retained more than 80% of its enzymatic and antigenic binding activities.     

Targeted protein functionalization using His-tags

With the impressive growth in gene sequence data that has become available, recombinant proteins represent an increasingly vast source of molecular components, with unique functional and structural properties, for use in biotechnological applications and devices. To facilitate the use, manipulation, and integration of such molecules into devices, a controllable method for their chemical modification was developed. In this approach, a trifunctional labeling reagent first recognizes and binds a His-tag on the target protein's surface. After binding, a photoreactive group on the trifunctional molecule is triggered to create a covalent linkage between the reagent and the target protein. The third moiety on the labeling reagent can be varied to bring unique chemical functionality to the target protein. This approach provides: (1) specificity in that only His-tagged targets are modified, (2) regio-specific control in that the target is modified proximal to the His-tag, the position of which can be varied, and (3) stoichiometric control in that the number modifications is limited by the binding capacity of the His-tag. Two such labeling reagents were designed, synthesized, and used to modify both N- and C-terminally His-tagged versions of the enzyme murine dihydrofolate reductase (mDHFR). The first reagent biotinylated the enzyme,while the second served to attach an oligonucleotide to yield a protein-DNA conjugate. In all cases, modification in this manner brings new functionality to the protein while leaving the enzymatic activity intact. The protein-DNA conjugate was used to specifically immobilize the active enzyme through DNA hybridization onto polystyrene microspheres, a step toward creating a functional protein microarray.     

Quantitation and characterization of the trifluoroacetonyl derivative of cysteine: a useful NMR probe

3-Bromo-1,1,1-trifluoropropanone (BrTFA) has been shown to be a sulfhydryl-specific reagent for proteins and peptides and, as such, a useful probe in protein NMR studies. This study describes two methods of quantitating the extent of sulfhydryl modification based on the stability of the γ,γ,γ-trifluoro-β-hydroxypropyl-cysteine derivative formed by the reduction of the γ,γ,γ-trifluoro-β-ketopropyl-cysteine group with NaBH₄. The proposed products are verified by NMR, and the quantitation methods are shown to be generally applicable to peptides and proteins.     

Kinetic Memory Based on the Enzyme-Limited Competition

Cellular memory, which allows cells to retain information from their environment, is important for a variety of cellular functions, such as adaptation to external stimuli, cell differentiation, and synaptic plasticity. Although posttranslational modifications have received much attention as a source of cellular memory, the mechanisms directing such alterations have not been fully uncovered. It may be possible to embed memory in multiple stable states in dynamical systems governing modifications. However, several experiments on modifications of proteins suggest long-term relaxation depending on experienced external conditions, without explicit switches over multi-stable states. As an alternative to a multistability memory scheme, we propose “kinetic memory” for epigenetic cellular memory, in which memory is stored as a slow-relaxation process far from a stable fixed state. Information from previous environmental exposure is retained as the long-term maintenance of a cellular state, rather than switches over fixed states. To demonstrate this kinetic memory, we study several models in which multimeric proteins undergo catalytic modifications (e.g., phosphorylation and methylation), and find that a slow relaxation process of the modification state, logarithmic in time, appears when the concentration of a catalyst (enzyme) involved in the modification reactions is lower than that of the substrates. Sharp transitions from a normal fast-relaxation phase into this slow-relaxation phase are revealed, and explained by enzyme-limited competition among modification reactions. The slow-relaxation process is confirmed by simulations of several models of catalytic reactions of protein modifications, and it enables the memorization of external stimuli, as its time course depends crucially on the history of the stimuli. This kinetic memory provides novel insight into a broad class of cellular memory and functions. In particular, applications for long-term potentiation are discussed, including dynamic modifications of calcium-calmodulin kinase II and cAMP-response element-binding protein essential for synaptic plasticity.     

Protein scaffolds can enhance the bistability of multisite phosphorylation systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large K(M), compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites.     

Influence of oxidation and crosslinking on oxygen binding properties of mouse erythrocytes

Different chemical treatments for mouse erythrocyte modification has been used. Oxidation treatments with Ascorbate/Fe(3+), a system able to react with intracellular proteins, produced a displacement of the O(2) binding equilibrium curve to a higher affinity behaviour with loss of the haemoglobin cooperativity for oxygen binding. Incubation of mouse erythrocytes with diamide showed that at low reagent concentration (0.8 mM) no modification on oxygen binding equilibrium curves was observed. At higher reagent concentration (2.0 mM), an increased affinity and a disappearance of the cooperative behaviour can be observed. Additionally, crosslinking reactions on mouse erythrocytes with band 3 crosslinkers seemed to affect oxygen binding properties when used at a crosslinker concentration of 5 mM. Oxyhaemoglobin levels in crosslinked and diamide-treated erythrocytes are similar to those found in control cells. In contrast, ascorbate/Fe(3+) treatments produced an increment in the proportion of methaemoglobin, decreasing the oxyhaemoglobin levels in these oxidized erythrocytes.     

Understanding glucose transport by the bacterial phosphoenolpyruvate:glycose phosphotransferase system on the basis of kinetic measurements in vitro

The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected. To address the issue of whether the behavior in vivo of the PTS can be understood in terms of these enzyme kinetics, a detailed kinetic model was constructed. Each overall phosphotransfer reaction was separated into two elementary reactions, the first entailing association of the phosphoryl donor and acceptor into a complex and the second entailing dissociation of the complex into dephosphorylated donor and phosphorylated acceptor. Literature data on the K(m) values and association constants of PTS proteins for their substrates, as well as equilibrium and rate constants for the overall phosphotransfer reactions, were related to the rate constants of the elementary steps in a set of equations; the rate constants could be calculated by solving these equations simultaneously. No kinetic parameters were fitted. As calculated by the model, the kinetic parameter values in vitro could describe experimental results in vivo when varying each of the PTS protein concentrations individually while keeping the other protein concentrations constant. Using the same kinetic constants, but adjusting the protein concentrations in the model to those present in cell-free extracts, the model could reproduce experiments in vitro analyzing the dependence of the flux on the total PTS protein concentration. For modeling conditions in vivo it was crucial that the PTS protein concentrations be implemented at their high in vivo values. The model suggests a new interpretation of results hitherto not understood; in vivo, the major fraction of the PTS proteins may exist as complexes with other PTS proteins or boundary metabolites, whereas in vitro, the fraction of complexed proteins is much smaller.     

Reactivity of cysteine-49 and its influence on the activation of microsomal glutathione transferase 1: evidence for subunit interaction

Microsomal glutathione transferase 1 is a homotrimeric detoxication enzyme protecting against electrophiles. The enzyme can also react with electrophiles, and when modification occurs at a unique Cys49 the reaction often results in activation. Here we describe the characterization of the chemical properties of this sulfhydryl (kinetic pK(a) was 8.8 +/- 0.3 and 9.0 +/- 0.1 with two different reagents) and we conclude that the protein environment does not lower the pK(a). Upon a direct comparison of the reactivity of Cys49 and low molecular weight thiols [L-Cys and glutathione (GSH)], the protein sulfhydryl displayed a 10-fold lower reactivity. The reactivity was correlated to reagent concentration in a linear fashion with a polar reagent, whereas the reactivity toward a hydrophobic reagent displayed saturation behavior (at low concentrations). This finding indicates that Cys49 is situated in a hydrophobic binding pocket. In a series of related quinones, activation occurs with the more reactive and less sterically hindered compounds. Thus, activation can be used to detect reactive intermediates during the metabolism of foreign compounds but certain intermediates can (and will) escape undetected. The reactivities of the three cysteines in the homotrimer were shown not to differ dramatically as the reaction of the protein with 4, 4'-dithiodipyridine could be fitted to a single exponential. On the basis of this result, a probabilistic expression could be used to relate the overall degree of modification to fractional activation. When N-ethylmaleimide activation (determined by the 1-chloro-2, 4-dinitrobenzene assay) was plotted against modification (determined with 4,4'-dithiodipyridine), a nonlinear relation was obtained, clearly showing that subunits do not function independently. The contribution to activation by single-, double-, and triple-modified trimers, were 0 +/- 0.06, 0.74 +/- 0.09, and 0.97 +/- 0.06, respectively. The double-modified enzyme appears partly activated, but this conclusion is more uncertain due to the possibility of independent modification of the purified enzyme upon storage. It is, however, clear that the single-modified enzyme is not activated whereas the triple-modified enzyme is fully activated. These observations together with the fact that MGST1 homotrimers bind only one substrate molecule (GSH) strongly support the view that subunits must interact in a functional manner.     

Post-translational modification of plant-made foreign proteins; glycosylation and beyond

The complex and diverse nature of the post-translational modification (PTM) of proteins represents an efficient and cost-effective mechanism for the exponential diversification of the genome. PTMs have been shown to affect almost every aspect of protein activity, including function, localisation, stability, and dynamic interactions with other molecules. Although many PTMs are evolutionarily conserved there are also important kingdom-specific modifications which should be considered when expressing recombinant proteins. Plants are gaining increasing acceptance as an expression system for recombinant proteins, particularly where eukaryotic-like PTMs are required. Glycosylation is the most extensively studied PTM of plant-made recombinant proteins. However, other types of protein processing and modification also occur which are important for the production of high quality recombinant protein, such as hydroxylation and lipidation. Plant and/or protein engineering approaches offer many opportunities to exploit PTM pathways allowing the molecular farmer to produce a humanised product with modifications functionally similar or identical to the native protein. Indeed, plants have demonstrated a high degree of tolerance to changes in PTM pathways allowing recombinant proteins to be modified in a specific and controlled manner, frequently resulting in a homogeneity of product which is currently unrivalled by alternative expression platforms. Whether a recombinant protein is intended for use as a scientific reagent, a cosmetic additive or as a pharmaceutical, PTMs through their presence and complexity, offer an extensive range of options for the rational design of humanised (biosimilar), enhanced (biobetter) or novel products.     

Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking

Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.     

Mathematical modeling of elution curves for a protein mixture in ion exchange chromatography applied to high protein concentration

Protein elution curves in ion exchange chromatography (IEC) were simulated with a rate model. Three pure proteins and their mixture were used (α‐lactalbumin, BSA, and conalbumin) under different operational conditions. The anionic matrix Q‐Sepharose FF was used packed in a 1 mL column. A high protein concentration (37.5 mg/mL of total protein injected into the column) was used in order to extend the utility of the model. Mass transfer parameters were calculated using empiric correlations, where the axial dispersion was negligible (Pe > 300) and the mass transfer was controlled by the intraparticle diffusion (Bi > 10). The model assumes a modulator–eluite relationship were the equilibrium constant of the Langmuir isotherm was a function of salt concentration. Adsorption kinetic parameters were estimated from experimental data. The parameters for pure proteins were determined, and elution curves for changes in flow rate, ionic strength gradient, concentration, and sample size were predicted by the model. Then the kinetic parameters of the mixture were determined under the same operational conditions and some of the parameters had to be modified to take into account effects such as protein–protein interactions, competition, and displacement. Experimental elution curves obtained for changes in operational conditions such as flow rate and ionic strength gradient were simulated by the rate model for the protein mixture with a relative error in retention time of visible peaks <5%. IEC operational conditions and the peak fraction collection can be selected using a cost function of the production process which considers yield, purity, concentration, and process time that are obtained from simulations. Operational conditions that gave the minimum cost were selected. Simulations allows to diminish experimental time and cost. Biotechnol. Bioeng. 2009; 104: 572–581 © 2009 Wiley Periodicals, Inc.