Efficient site-specific labeling of proteins via cysteines

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.     

Cooperativity of the oxidization of cysteines in globular proteins

Based on the 639 non-homologous proteins with 2910 cysteine-containing segments of well-resolved three-dimensional structures, a novel approach has been proposed to predict the disulfide-bonding state of cysteines in proteins by constructing a two-stage classifier combining a first global linear discriminator based on their amino acid composition and a second local support vector machine classifier. The overall prediction accuracy of this hybrid classifier for the disulfide-bonding state of cysteines in proteins has scored 84.1% and 80.1%, when measured on cysteine and protein basis using the rigorous jack-knife procedure, respectively. It shows that whether cysteines should form disulfide bonds depends not only on the global structural features of proteins but also on the local sequence environment of proteins. The result demonstrates the applicability of this novel method and provides comparable prediction performance compared with existing methods for the prediction of the oxidation states of cysteines in proteins.     

Mustard gas crosslinking of proteins through preferential alkylation of cysteines

Mustard gas,bis(2-chloroethyl)sulfide, treatment of proteins is shown to generate significant amounts of covalently crosslinked protein dimers. This is due to the preferential alkylation of cysteine residues. Crosslinking does not occur in the model protein staphylococcal nuclease, which has no cysteine residues. Treatment of cysteine-containing mutants of staphylococcal nuclease with this chemical warfare agent did result in crosslinking. However, these dimers are slowly cleaved back to monomers by an unknown mechanism. The alkylation and crosslinking of cysteine-containing proteins by mustard gas may contribute to its toxicity.     

Amino acid neighbours and detailed conformational analysis of cysteines in proteins.

Here we present an investigation of the contacts that cysteines make with residues in their three-dimensional environment and a comprehensive analysis of the conformational features of 351 disulphide bridges in 131 non-homologous single-chain protein structures. Upstream half-cystines preferentially have downstream neighbours, whereas downstream half-cystines have mainly upstream neighbours. Non-disulphide bridged cysteines (free cysteines) have no preference for upstream or downstream neighbours. Free cysteines have more contacts to non-polar residues and fewer contacts to polar/charged residues than half-cystines, which correlates with our observation that free cysteines are more buried than half-cystines. Free cysteines prefer to be located in alpha-helices while no clear preference is observed for half-cystines. Histidine and methionine are preferentially seen nearby free cysteines. Tryptophan is found preferentially nearby half-cystines. We have merged sequential and spatial information, and highly interesting novel patterns have been discovered. The number of cysteines per protein is typically an even number, peaking at four. The number of residues separating two half-cystines is preferentially 11 and 16. Left-handed and right-handed disulphide bridges display different conformational parameters. Here we present side chain torsion angle information based on a 5-12 times larger number of disulphide bridges than has previously been published. Considering the importance of cysteines for maintaining the 3D-structural scaffold of proteins, it is essential to have as accurate information as possible concerning the packing and conformational preferences. The present work may provide key information for engineering the protein environment around cysteines.     

An assay to quantitate reducible cysteines from nanograms of GST-fusion proteins

Cysteine residues in proteins are targets of numerous post-translational modifications and play important roles in protein structure and enzymatic function. Consequently, understanding the full biochemistry of proteins depends on determining the oxidation state and availability of the residues to be modified. We have developed a highly sensitive assay that accurately determines the number of unmodified cysteine residues in GST-fusion proteins. Only picomoles of protein are required for each reaction, which are carried out in 96-well glutathione-coated plates. Free unmodified cysteine residues are labeled and quantified using biotin and HRP-conjugated streptavidin. Our assay can be used to quantify reactions targeting sulfhydryl groups in proteins. We demonstrate this assay using full-length and truncation mutants of the SNARE proteins syntaxin1A, SNAP-25B, and synaptobrevin2, which have 0–4 cysteines. We are able to accurately determine the number of cysteine residues in each protein and follow the modification of these cysteines by oxidation and reaction with NEM (N-ethylmaleimide). This assay is as simple as running an ELISA or Western blot and, because of its high resolution, should allow detailed analysis of the chemistry of cysteine residues in proteins.     

Prediction of the disulfide-bonding state of cysteines in proteins at 88% accuracy

The task of predicting the cysteine-bonding state in proteins starting from the residue chain is addressed by implementing a new hybrid system that combines a neural network and a hidden Markov model (hidden neural network). Training is performed using 4136 cysteine-containing segments extracted from 969 nonhomologous proteins of well-resolved three-dimensional structure. After a 20-fold cross-validation procedure, the efficiency of the prediction scores as high as 88% and 84%, when measured on cysteine and protein basis, respectively. These results outperform previously described methods for the same task.     

Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex

Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cellulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1Cel6A) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted Z(SPA-1)), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1Cel6A fusion protein had been previously applied. This showed that the SPA-CBM1Cel6A fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the Z(SPA-1) affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.     

Maximal stabilities of reversible two-state proteins

The hydrophobic effect is the major force driving protein folding. Around room temperature, small organic solutes and hydrophobic amino acids have low solubilities in water and the hydrophobic effect is the strongest. These facts suggest that globular proteins should be maximally stable around room temperature. While this fundamental paradigm has been expected, it has not actually been shown to hold. Toward this goal, we have collected and analyzed experimental thermodynamic data for 31 proteins that show reversible two-state folding <--> unfolding transitions at or near neutral pH. Twenty-six of these are unique, and 20 of the 26 are maximally stable around room temperature irrespective of their structural properties, the melting temperature, or the living temperatures of their source organisms. Their average temperature of maximal stability is 293 +/- 8 K (20 +/- 8 degrees C). These proteins differ in size, fold, and number of domains, hydrophobic folding units, and oligomeric states. They derive from the cold-loving psychrophiles, from mesophiles, and from thermophiles. Analysis of the single-domain proteins present in this set shows that the variations in their thermodynamic parameters are correlated in a way which may explain the adaptation of the proteins to the living temperatures of the organisms from which they derive. The average energetic contribution of the individual amino acids toward protein stability decreases with an increase in protein size, suggesting that there may be an upper limit for protein maximal thermodynamic stability. For the remaining proteins, deviation of the maximal stability temperatures from room temperature may be due to greater uncertainties in their heat capacity change (DeltaC(p)) values, a weaker hydrophobic effect, and/or a stronger electrostatic contribution.     

Cell-type specific processing of neuroendocrine precursor proteins using vaccinia recombinants

The cell-type specific processing of human pro-enkephalin was determined using a vaccinia recombinant (VV:hPE). The results show that all cell types infected with VV:hPE efficiently synthesize pro-enkephalin following cleavage of the signal peptide after Ala24. In addition, pro-enkephalin is shown to undergo N-linked glycosylation as well as other post-translational modifications. However, only one cell line. AtT-20, was able to efficiently cleave pro-enkephalin to smaller peptides including Met-enkephalin. Some results have been previously reported (Science 232, 1641-1643).     

Isolation of specific RNA-binding proteins using the streptomycin-binding RNA aptamer

Here we report a simple and cheap one-step affinity purification protocol for isolating RNAs or proteins that interact with selected functional RNAs. The streptomycin-binding aptamer, termed 'StreptoTag,' is embedded in or fused to either end of any RNA of interest. The resulting hybrid RNA can then be immobilized on a streptomycin affinity matrix. When a complex protein mixture or total cellular lysate is applied to the matrix, subsequent elution with free streptomycin allows efficient recovery of specific ribonucleoprotein or RNA-RNA complexes. The method was successfully used to purify yeast and phage RNA-binding proteins and group II intron, viral and bacterial noncoding RNA (ncRNA)-binding proteins. The selective enrichment of bacterial mRNAs that bind ncRNAs has also been demonstrated. Once the affinity matrix, the RNA construct and the protein extracts have been prepared, the experimental procedure can be performed in 1-2 h.     

Free energy of charges in solvated proteins: microscopic calculations using a reversible charging process

Evaluation of the free energy of ionization of acidic groups in proteins may be used as a powerful and general test case for determining the reliability of calculations of electrostatic energies in macromolecules. This work attacks this test case by using an adiabatic charging process that evaluates the changes in free energies associated with ionizing the acidic groups Asp-3 and Glu-7 in bovine pancreatic trypsin inhibitor and aspartic acid in solution. The results of these free energy calculations are very encouraging; the error range is about 1 kcal/mol for these free energy changes of about-70 kcal/mol. This indicates that we are finally approaching the stage of obtaining quantitative results in modeling the energetics of solvated proteins.     

Labeling proteins with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate

Two methods were investigated for the no-carrier-added synthesis of N-succinimidyl 4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB). The first, an attempted nucleophilic aromatic substitution by [¹⁸F]fluoride on N-succinimidyl 4-nitrobenzoate was unsuccessful. The second method involved three steps; [¹⁸F]fluoride for trimethylammonium substitution on 4-formyl-N,N,N-trimethylanilinium triflate, oxidation to 4-[¹⁸F]fluorobenzoic acid, followed by reaction with N-hydroxysuccinimide and dicyclohexylcarbodiimide to form S[¹⁸F]FB. Total synthesis and purification time was 100 min and the overall radiochemical yield was 25% (decay corrected). A monoclonal antibody F(ab′)₂ fragment could be labeled in 40–60% yield by reaction with S[¹⁸F]FB for 15–20 min. The tissue distribution in normal mice and in vitro tumor binding of the antibody F(ab′)₂ labeled by reaction with S[¹⁸F]FB were comparable to those observed for the fragment after radioiodination using N-succinimidyl 4-[¹²⁵I]iodobenzoate.     

Labeling of proteins using [105Rh]Rh-4-(p-aminobenzyl)-diethylenetriamine

A bifunctional ligand 4-(p-aminobenzyl)-diethylenetriamine has been synthesized. ¹⁰⁵Rh complexes with this ligand were prepared with an overall yield of 79% at pH 9.0 in bicarbonate buffer. The preformed complex was converted to the isothiocyanate derivative using thiophosgene. Conjugation yields of 75% with IgG and 85% with HSA could be obtained for a 4 h conjugation reaction. Affinity chromatography of human-IgG coupled to the rhodium complex in an anti-human IgG agarose gel indicated no denaturation of the labeled protein. The procedure reported here can be adapted for the preparation of ¹⁰⁵Rh-labeled antibodies for therapeutic applications.     

Study of human chromosomes. IV. Labeling of chromosomal proteins with the amino group specific fluorescent reagent fluorescamine

We describe a method for labeling chromosomal proteins with an amino-group-specific fluorescent reagent, fluorescamine. Chromosomes thus labeled appear either as uniformly fluorescent or as haloes in structure depending on the proteins remaining after treatment with acid-alcohol fixation. Using fluorescamine as a probe, we demonstrate that there is a substantial loss of labeled proteins during the chromosomal preparation and also during the trypsin treatment used in the banding of chromosomes.     

Prediction of the disulfide bonding state of cysteines in proteins with hidden neural networks

A hybrid system (hidden neural network) based on a hidden Markov model (HMM) and neural networks (NN) was trained to predict the bonding states of cysteines in proteins starting from the residue chains. Training was performed using 4136 cysteine-containing segments extracted from 969 non-homologous proteins of well-resolved 3D structure and without chain-breaks. After a 20-fold cross-validation procedure, the efficiency of the prediction scores as high as 80% using neural networks based on evolutionary information. When the whole protein is taken into account by means of an HMM, a hybrid system is generated, whose emission probabilities are computed using the NN output (hidden neural networks). In this case, the predictor accuracy increases up to 88%. Further, when tested on a protein basis, the hybrid system can correctly predict 84% of the chains in the data set, with a gain of at least 27% over the NN predictor.     

Proteomic profile of reversible protein oxidation using PROP, purification of reversibly oxidized proteins

Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells.