Absolute Quantification of Prion Protein (90–231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrP(TSE)) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrP(TSE) has the ability to convert the conformation of the normal prion protein (PrP(C)) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrP(TSE) at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrP(TSE), the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.     

Fragment-Based Stabilizers of Protein–Protein Interactions through Imine-Based Tethering

Small-molecule stabilization of protein–protein interactions (PPIs) is a promising concept in drug discovery, however the question how to identify or design chemical starting points in a “bottom-up” approach is largely unanswered. We report a novel concept for identifying initial chemical matter for PPI stabilization based on imine-forming fragments. The imine bond offers a covalent anchor for site-directed fragment targeting, whereas its transient nature enables efficient analysis of structure–activity relationships. This bond enables fragment identification and optimisation using protein crystallography. We report novel fragments that bind specifically to a lysine at the PPI interface of the p65-subunit-derived peptide of NF-κB with the adapter protein 14-3-3. Those fragments that subsequently establish contacts with the p65-derived peptide, rather than with 14-3-3, efficiently stabilize the 14-3-3/p65 complex and offer novel starting points for molecular glues.     

Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein–Protein Interactions

In the present work, we proposed to create special sorbents for the study of protein–protein interactions, based on the fixation of cysteine-inserted beta-casein mutants with thiol-Sepharose resin. As a model system, we used bovine beta-casein, which belongs to the family of intrinsically unstructured proteins. Insertion of distal cysteines into the unfolded protein was not found to significantly change beta-casein properties. An amphiphilic beta-casein molecule has one hydrophilic domain and one hydrophobic domain placed on the N- and C-terminus, thus enabling one to exploit its capacity to engage in different types of intermolecular interactions. Two different casein-Sepharose sorbents incorporating either C-4 or C-208 beta-casein mutants bound to thiol-Sepharose were produced, exposing the hydrophobic domain in the case of the C-4 and the hydrophilic domain in the case of the C-208 mutant, respectively. The results obtained using the proposed sorbents with native beta-casein, another partially unfolded protein prion, and an oligomeric globular glyceraldehyde-3-phosphate dehydrogenase were found to be consistent with the data obtained by ELISA on free protein–protein complexes. Thus, Sepharose modified with various proteins is suitable for isolation of proteins interacting with the chromatographic phase bound partners from multicomponent systems such as milk. The obtained results allow the proposing of a fast and convenient method to be used for isolation of proteins, determination of protein-interacting partners, and the study of multi-protein complexes.     

Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein–Protein Interactions

In the present work, we proposed to create special sorbents for the study of protein–protein interactions, based on the fixation of cysteine-inserted beta-casein mutants with thiol-Sepharose resin. As a model system, we used bovine beta-casein, which belongs to the family of intrinsically unstructured proteins. Insertion of distal cysteines into the unfolded protein was not found to significantly change beta-casein properties. An amphiphilic beta-casein molecule has one hydrophilic domain and one hydrophobic domain placed on the N- and C-terminus, thus enabling one to exploit its capacity to engage in different types of intermolecular interactions. Two different casein-Sepharose sorbents incorporating either C-4 or C-208 beta-casein mutants bound to thiol-Sepharose were produced, exposing the hydrophobic domain in the case of the C-4 and the hydrophilic domain in the case of the C-208 mutant, respectively. The results obtained using the proposed sorbents with native beta-casein, another partially unfolded protein prion, and an oligomeric globular glyceraldehyde-3-phosphate dehydrogenase were found to be consistent with the data obtained by ELISA on free protein–protein complexes. Thus, Sepharose modified with various proteins is suitable for isolation of proteins interacting with the chromatographic phase bound partners from multicomponent systems such as milk. The obtained results allow the proposing of a fast and convenient method to be used for isolation of proteins, determination of protein-interacting partners, and the study of multi-protein complexes.

     

Multicomponent Peptide Stapling as a Diversity-Driven Tool for the Development of Inhibitors of Protein–Protein Interactions

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein–protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction-based stapling is an effective strategy for the development of α-helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo- and endo-cyclic hydrophobic moieties at the side chain cross-linkers. The interaction of the Ugi-staple fragments with the target protein was demonstrated by crystallography.     

Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides provides powerful insight into the quaternary structure of protein complexes. Mixed-isotope cross-linking (a method for distinguishing intermolecular cross-links) was coupled with liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) to provide an additional separation dimension to the traditional cross-linking approach. This method produced multiplet m/z peaks that are aligned in the IMS drift time dimension and serve as signatures of intermolecular cross-linked peptides. We developed an informatics tool to use the amino acid sequence information inherent in the multiplet spacing for accurate identification of the cross-linked peptides. Because of the separation of cross-linked and non-cross-linked peptides in drift time, our LC-IMS-MS approach was able to confidently detect more intermolecular cross-linked peptides than LC-MS alone.      

Prion疾病与生物无机化学

介绍了除病菌、病毒以外的第三种病原体－－病蛋白Ｐｒｉｏｎ。这种病原蛋白的本质是正常蛋白的异常折叠,因此又被称为蛋白分子的“构象病”。还介绍了这种病蛋白的可能致病机理。铜离子对这种病蛋白生成的重要作用,充分显示了生物无机化学这门新兴学科的重要性和前沿性。     

Advances in Detection Methods of β-Amyloid Protein

Alzheimer's disease (AD) is a neurodegenerative disease with unknown etiology. β-amyloid protein (Aβ) is one of the specific biomarkers of AD, and many clinical studies suggest that abnormal levels of Aβ in blood, cerebrospinal fluid and brain tissue are closely related to the progression of AD. The analysis and evaluation of Aβ are important for early detection, tracking, prevention, and treatment of AD. In this paper, the present situations of the commonly used detection methods of Aβ at home and abroad were summarized and compared. Specifically, the latest application of new electrochemical biosensor in Aβ detection was mainly described, and the summary of its future directions and the potential applications was given.     

Purification and Characterization of PRL Protein Tyrosine Phosphatases

PRLs constitute a subfamily of protein tyrosine phosphatases(PTPs). In the present paper are reported the molecular cloning, expression, purification, and characterization of all the three members of the PRL enzymes in human and the only PRL in C. elegans. These enzymes were expressed as glutathione S-transferase (GST) fusion proteins in DE3pLysS E. coil cells, and the recombinant fusion proteins were purified on glutathione-Sepharose affinity columns. Having been cleaved with thrombin, GST-free enzymes were further purified on an S-100 Sepharose gel filtration column. The purified proteins show single polypeptide bands on SDS-polyacrylamide gel electrophoresis. With para-nitrophenyl phosphate(p-NPP) as a substrate, PRLs exhibit classical Michaelis-Menten kinetics with Vmax values two orders of magnitude smaller than those of classic PTPs. The responses of PRLs to ionic strength, metal ions and phosphatase inhibitors are similar to those of other characterized PTPs, but their optimal pH values are different. These data thus reveal distinct common biochemical properties of PRL subfamily PTPs as well.     

Influence of Sulfolane on ESI-MS Measurements of Protein–Ligand Affinities

The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein–ligand affinities are described. Binding measurements carried out on four protein–carbohydrate complexes, lysozyme with β-d-GlcNAc-(1→4)-β-d-GlcNAc-(1→4)-β-d-GlcNAc-(1→4)-d-GlcNAc, a single chain variable fragment and α-d-Gal-(1→2)-[α-d-Abe-(1→3)]-α-d-Man-OCH₃, cholera toxin B subunit homopentamer with β-d-Gal-(1→3)-β-d-GalNAc-(1→4)[α-d-Neu5Ac-(2→3)]-β-d-Gal-(1→4)-β-d-Glc, and a fragment of galectin 3 and α-l-Fuc-(1→2)-β-d-Gal-(1→3)-β-d-GlcNAc-(1→3)-β-d-Gal-(1→4)-β-d-Glc, revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein–ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme–tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.

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A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein–Protein Conjugates

Bioorthogonal chemistry holds great potential to generate difficult-to-access protein–protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DA_inv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin-based targeted cancer therapies.     

Structure Analysis of Streptococcal Protein G Fc Binding Domain

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant     

Probability Properties of Multi－contact in Protein Molecules

The compact conformations of polymers are important because the native conformations of all bio-polymers with certain function are highly compact. The properties of mutil-contact bio-polymer chains were studied by Gaussian statistics of the random-flight chain. The theoretical expressions(were given, also), the calculations of probability distributions and correlation functions for different topologic cases were derived and made respectively. Comparison between single, double and triple contacts was also made. By means of setting the parameters, the results of the current calculations of the multiple contacts are just the same as those calculated by single, double or tripe contacts separately. It is a useful method to investigate native conformations of biopolymers. The probabilities of multi contacts and correlation functions between chain‘s contacts were calculated for the Gaussian chains. Because the bond probability distributions are Gaussian‘s distributions, the probability distributions of the separations of various points along the chains are always consecutive. All the contacts may break up into several groups, and each group consists of many contacts. Here we investigated the probability distribution from one group to three groups of contacts.     

A Cooperative Approach for the Extraction of Protein Motifs

Many problems in chemistry depend on the ability to identify the global solution of a function, which can be a minimum or a maximum. Because the number of local optima grows exponentially with the complexity of the problems, finding the global optimum tur…     

Development of New Capillary Electrophoresis-Based Techniques for Protein Analysis

With the sequencing of human genome almost complete, human genome project enters the postgenome-sequencing era. Compared to genomics, the analysis of proteome is rather difficult since in human cells there are around 200 000 proteins, which are expressed at any time at different levels     

Probing the Selectivity and Protein⋅Protein Interactions of a Nonreducing Fungal Polyketide Synthase Using Mechanism-Based Crosslinkers

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Recognition of Protein Native Folds Using a Continuous Potential Function

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Development of a Protein Chip to Measure PKCβ Activity

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C β (PKCβ) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCβ activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCβ was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCβ substrate peptide on epoxy-treated slide surface, PKCβ reaction mix was applied over the immobilized MBP-fused PKCβ substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCβ protein chip as a high-throughput screening tool in the screening of depigmenting agents.     

Initiation of Protein Synthesis with Non-Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.