Recent advances of thiol-selective bioconjugation reactions

Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes, fulfilling specific functions with unmatched precision. This unique specificity makes proteins an ideal scaffold to generate tools for the exploration of natural systems or for the construction of modern therapeutics. Thus, the chemoselective modification of proteins with functionalities that are not defined by the genetic code has become an indispensable approach for life science research and the development of therapeutics. Amongst site-selective strategies for protein modification, cysteine-selective approaches have long been used for the generation of functional protein conjugates and new reactions continue to emerge, offering solutions for diverse research questions. In this review, we are highlighting new strategies for the chemoselective modification of cysteine residues in peptides, proteins and antibodies with a particular focus on the most recent years. We lay special focus on new reagents for efficient cysteine conjugation that produce stable conjugation products with significant pharmaceutical application.     

Oxidation of carboxyamidomethyl cysteine may add complexity to protein identification

Protein identification by interrogation of databases requires a comprehensive compilation of modified amino acids forms. Here, we describe the chemical oxidation of carboxyamidomethyl cysteine to the sulfoxide and sulfone forms, species that may add more complexity to peptide analyses. They can be easily distinguished by tandem mass spectrometry (MS/MS) due to their characteristic pattern of side chain neutral eliminations either from the parent ion or ion series that generate dehydroalanine as detected by MS(3). This finding was supported by the MS(n) spectra recorded for a peptide isolated from a mixture of tryptic peptides and for a derivatized/oxidized synthetic peptide with a different sequence. These modifications and their diagnostic neutral losses should be included in the list of chemical modifications and in algorithms designed for the automatic sequencing of peptides and database searching.     

Amino Acid Biases in the N- and C-termini of Proteins are Evolutionarily Conserved and are Conserved Between Functionally Related Proteins

Analysis of the Arabidopsis thaliana, Saccharomyces cerevisiae, Mus musculus, Escherichia coli, Bacillus subtilis, Thermoplasma acidophilum, and Sulfolobus tokodaii genomes demonstrate that many amino acid biases occur at the N- and C-termini of proteins, a statistically significant number of these biases are evolutionarily conserved, and these biases occur in amino acids beyond the first and last five amino acids. Analyses designed to shed light on the mechanism causing amino acid biases suggest that in at least some cases the bias is caused by forces acting at the nucleic acid level. It is also demonstrated that in E. coli functionally related proteins show similar biases at the N- and C-termini suggesting that the mechanisms causing the biases are complex and in some cases are related to function.     

5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2

Structural studies of human chloride intracellular channel protein 2 (CLIC2) had been hampered by the problem of generating suitable crystals primarily due to the protein containing exposed cysteines. Several chemical reagents were used to react with the cysteines on CLIC2 in order to modify the redox state of the protein. We have obtained high quality crystals that diffracted to better than 2.5 Å at a home X-ray source by treating the protein with 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB). After solving the crystal structure of CLIC2, we found that the DTNB had reacted with the Cys¹¹⁴, and made CLIC2 in a homogenous oxidized state. This study demonstrated that the DTNB modification drastically improved the crystallization of CLIC2, and it implied that this method may be useful for other proteins containing exposed cysteines in general.     

Recent advances in mammalian protein production

Mammalian protein production platforms have had a profound impact in many areas of basic and applied research, and an increasing number of blockbuster drugs are recombinant mammalian proteins. With global sales of these drugs exceeding US$120 billion per year, both industry and academic research groups continue to develop cost effective methods for producing mammalian proteins to support pre-clinical and clinical evaluations of potential therapeutics. While a wide range of platforms have been successfully exploited for laboratory use, the bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins. In this review we highlight the range of mammalian expression platforms available for recombinant protein production, as well as advances in technologies for the rapid and efficient selection of highly productive clones.     

羧酸还原酶的研究进展

羧酸还原酶(carboxylic acid reductases,CARs)可以催化羧酸还原为相应的醛,反应条件温和、拥有广阔的底物范围且副反应较少。本文旨在综述近年来羧酸还原酶系统发育、结构与催化机理、蛋白质工程和固定化工程等方面的研究成果,揭示其作为重要工具酶在生物转化及合成生物学中的应用前景。     

On the cysteine and cystine content of proteins

Summary Analysis of published data on the cysteine and half-cystine content of proteins indicates that most intracellular proteins may be classified as sulfhydryl proteins (those containing cysteine but little or no half-cystine) and that such sulf-hydryl proteins have a low cysteine content. The mean cysteine content found for 32 intracellular mammalian proteins was 1.6 % and intracellular proteins of many bacteria have similar or lower values. Extracellular mammalian proteins are primarily disulfide proteins (those containing half-cystine but little or no cysteine) and have a high half-cystine content, the mean value found for some 34 extracellular mammalian proteins being 4.1 %. This is contrasted with many of the extracellular proteins from facultative bacteria which are cyst(e)ine-free proteins, being lacking in both cysteine and half-cystine. These and related observations are interpreted in terms of the evolution of life in a reducing atmosphere and the subsequent transition to an oxidizing environment. It is suggested that disulfide proteins evolved primarily after the accumulation of oxygen in the atmosphere.     

Improvement of translation efficiency in an Escherichia coli cell-free protein system using cysteine

Cell-free protein synthesis systems are powerful tools for protein expression, and allow large amounts of specific proteins to be obtained even if these proteins are detrimental to cell survival. In this report we describe the effect of cysteine on cell-free protein synthesis. The addition of cysteine caused a 2.7-fold increase in the level of synthesized glutathione S-transferase (GST). Moreover, the levels of sulfhydryl group reductants, including reduced glutathione and dithiothreitol (DTT), were increased 1.9- and 1.7-fold, respectively, whereas levels of the disulfide dimers, cystine and oxidized glutathione, were suppressed 87% and 66%, respectively. These trends were also observed for green fluorescent protein (GFP) expression. The addition of cysteine competitively reversed the inhibitory effect of cystine on protein expression. These results suggest that the sulfhydryl group in cysteine plays a crucial role in enhancing protein synthesis, and that the addition of excess cysteine could be a convenient and useful method for improving protein expression.     

Improved protein sequence coverage by on resin deglycosylation and cysteine modification for biomarker discovery

Membrane proteins and secreted factors (soluble proteins or extracellular matrix components) are the targets of most monoclonal antibodies, which are currently in clinical development. These proteins are frequently post‐translationally modified, e.g. by the formation of disulfide bonds or by glycosylation, which complicates their identification using proteomics technologies. Here, we describe a novel methodology for the on resin deglycosylation and cysteine modification of proteins after in vitro, in vivo or ex vivo biotinylation. Biotinylated proteins are captured on streptavidin resin and all subsequent modifications, as well as the proteolytic digestion, which yields peptides for MS analysis, are performed on resin. Using biotinylated bovine fetuin‐A as a test protein, an improvement in sequence coverage from 7.9 to 58.7% could be shown, including the identification of all three glycosylation sites. Furthermore, a complex mixture derived from the ex vivo biotinylation of vascular structures in human kidney with cancer obtained by perfusion after surgical resection revealed almost a doubling of sequence coverage for all checked proteins when analyzed by LC‐MALDI TOF/TOF.     

Large scale modification of biomolecules using immobilized sortase A from Staphylococcus aureus

Recently, sortase A (SrtA) from Staphyloccus aureus moved into the focus of bioscience because of its ability to incorporate site specific modifications into proteins. The enzyme was mostly used to modify target proteins in an analytical scale, to study biomolecules in their cellular context. In this study, we show the applicability of SrtA mediated ligation for site specific modification of proteins in a large scale. Therefore, the reaction was first optimized using peptides and subsequently new reaction conditions were applied for the large scale biotinylation of interleukin-8. Furthermore, we established C-terminal immobilization of the SrtA on a PEG based resin and could demonstrate maintaining enzymatic activity. Immobilized SrtA significantly facilitates previous ligation protocols as the enzyme can be easily recycled. Also, the removal of excess reaction solution and the whole washing process is significantly accelerated, as centrifugation or filtration techniques can be applied instead of time-consuming chromatography steps.     

Importance of N- and C-terminal regions of IbpA, Escherichia coli small heat shock protein, for chaperone function and oligomerization

Small heat shock proteins are ubiquitous molecular chaperones that, during cellular stress, bind to misfolded proteins and maintain them in a refolding competent state. Two members of the small heat shock protein family, IbpA and IbpB, are present in Escherichia coli. Despite 48% sequence identity, the proteins have distinct activities in promoting protein disaggregation. Cooperation between IbpA and IbpB is crucial for prevention of the irreversible aggregation of proteins. In this study, we investigated the importance of the N- and C-terminal regions of IbpA for self-oligomerization and chaperone functions. Deletion of either the N- or C-terminal region of IbpA resulted in a defect in the IbpA fibril formation process. The deletions also impaired IbpA chaperone function, defined as the ability to stabilize, in cooperation with IbpB, protein aggregates in a disaggregation-competent state. Our results show that the defect in chaperone function, observed in truncated versions of IbpA, is due to the inability of these proteins to interact with substrate proteins and consequently to change the properties of aggregates. At the same time, these versions of IbpA interact with IbpB similarly to the wild type protein. Competition experiments performed with the pC peptide, which corresponds to the IbpA C terminus, suggested the importance of IbpA intermolecular interactions in the stabilization of aggregates in a state competent for disaggregation. Our results suggest that these interactions are not only dependent on the universally conserved IEI motif but also on arginine 133 neighboring the IEI motif. IbpA mutated at arginine 133 to alanine lacked chaperone activity.     

Analysis of the stability and function of nucleoplasmin through cysteine mutants

Xenopus laevis nucleoplasmin is a pentameric nuclear chaperone. The relation between the structure and the multifunctional aspects of the molecule has not yet been clearly established. In the course of analysing a C-terminally His-tagged recombinant version of the region equivalent to the trypsin resistant core (r-NP142) of the molecule, we found that this domain exhibited a substantially decreased oligomerization potential. To better understand the role of the three cysteines of nucleoplasmin on its pentameric functional structure, we have selectively mutated these residues to serine and generated three mutants (C15S, C35S, and C45S) both for the complete recombinant nucleoplasmin (r-NP) and the truncated r-NP142 non-tagged forms. We demonstrate that there are no disulphide bridges stabilizing either the monomer or the pentamer. Neither C15S nor C35S has any structural effects, while the mutation C45S abolishes the ability of r-NP142 to pentamerize. This structural impairment suggests that hydrophobic interactions of Cys 45 are critical for the stability of the protein. Our studies allow to analyse for the first time the structural and functional properties of nucleoplasmin in its monomeric form.     

Analysis of cysteine residues in peptides and proteins alkylated with volatile reagents

Summary Conditions are described for the reduction and alkylation of cysteines in peptides and proteins with volatile reagents by use of triethylphosphine as reductant, bromopropane as alkylating reagent and triethylamine as base. Alkylated samples need only be vacuum dried prior to subsequent analysis steps. Alkylated samples have been acid hydrolyzed and analyzed on an amino acid analyzer with recoveries of cysteine within 10% of the expected value. Alkylated samples have been directly applied to a sequencer membrane, dried on the surface and cysteines identified by sequence analysis without additional wash steps. In addition proteins blotted onto PVDF have been alkylatedin situ and sequenced with identification of cysteines. On the analyzer and sequencer the S-propylcysteine derivative elutes at a unique position allowing for the unambiguous identification of cysteine. Cysteine residues are quantitativly alkylated under the conditions developed. The ease of this procedure allows the routine analysis of cysteine in peptides and proteins without additional, time consuming repurification or dialysis steps.Abbreviations dptu diphenylthiourea - dmptu dimethylphenylthiourea - prop-cys S-propylcysteine     

The impact of single cysteine residue mutations on the replication terminator protein

We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies. The cysteine residue 110 was replaced with alanine, valine or serine. Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP. The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity. The structure of RTP.C110S, solved to 2.5A resolution using crystallographic methods, showed no major structural perturbation due to the mutation. Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation. The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.     

Mapping protein cysteine sulfonic acid modifications with specific enrichment and mass spectrometry: An integrated approach to explore the cysteine oxidation

Oxidation of thiol proteins, which results in conversion of cysteine residues to cysteine sulfenic, sulfinic or sulfonic acids, is an important posttranslational control of protein function in cells. To facilitate the analysis of this process with MALDI‐MS, we have developed a method for selective enrichment and identification of peptides containing cysteine sulfonic acid (sulfopeptides) in tryptic digests of proteins based on ionic affinity capture using polyarginine‐coated nanodiamonds as high‐affinity probes. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a complex peptide mixture in which the abundance of the sulfonated analyte is as low as 0.02%. The polyarginine‐coated probes exhibit a higher affinity for peptides containing multiple sulfonic acids than peptides containing single sulfonic acid. The limit of the detection is in the femtomole range, with the MALDI‐TOF mass spectrometer operating in the negative ion mode. The results show that the new approach has good specificity even in the presence of phosphopeptides. An application of this method for selective enrichment and structural identification of sulfopeptides is demonstrated with the tryptic digests of performic‐acid‐oxidized BSA.     

Characterization of cysteine string protein in rat parotid acinar cells

Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70 kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP1_1–112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP1_1–82, containing the J-domain only, nor GST-CSP1_83–112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.     

Enhancing long-term thermal stability in mesophilic glutamate dehydrogenase from Clostridium symbiosum by eliminating cysteine residues

Glutamate dehydrogenase from Clostridium symbiosum has two cysteine residues, C144 and C320. The single mutant C320S and a double mutant with both cysteines replaced by serine have been compared with one another in terms of long-term stability and other properties. Specific activities and kinetic parameters were relatively little affected, but stability was improved—e.g. at 25 °C sterile, sealed samples of wild-type enzyme, C320S and the double mutant at 0.1 mg/ml in 0.1 M phosphate buffer, pH 7 lost 50%, 42% and 32% of activity over 60 days. For the first two proteins this loss was partly reversible with dithiothreitol. When wild-type enzyme was deliberately contaminated with 1 μM Cu²⁺ it became less stable and formed aggregates, whereas the double mutant was not affected. The double mutation thus removes a source of instability through –SH oxidation that would be accentuated by any heavy metal contamination of solutions.     

Recent advances in protein methylation: Enzymatic methylation of nucleic acid binding proteins

Summary Heterogeneous nuclear RNP protein A1, one of the major proteins in hnRNP particle (precursor for mRNA), is known to be post-translationally arginine-methylatedin vivo on residues 193, 205, 217 and 224 within the RGG box, the motif postulated to be an RNA binding domain. Possible effect of N^G-arginine methyl-modification in the interaction of protein A1 to nucleic acid was investigated. The recombinant hnRNP protein A1 wasin vitro methylated by the purified nuclear protein/histone-specific protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase) stoichiometrically and the relative binding affinity of the methylated and the unmethylated protein A1 to nucleic acid was compared: Differences in their binding properties to ssDNA-cellulose, pI values and trypsin sensitivities in the presence and absence of MS2-RNA all indicate that the binding property of hnRNP protein A1 to single-stranded nucleic acid has been significantly reduced subsequent to the methylation. These results suggest that posttranslational methyl group insertion to the arginine residue reduces protein-RNA interaction, perhaps due to interference of H-bonding between guanidino nitrogen arginine and phosphate RNA.Abbreviations hnRNP heterogeneous ribonucleoprotein particle - AdoMet S-adenosyl-L-methionine - AdoHcy S-adenosyl-L-homocysteine - MBP myelin basic protein - HMG high mobility group - ss single stranded     

A helical conotoxin from Conus imperialis has a novel cysteine framework and defines a new superfamily

Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily. The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified.