A modular cross-linking approach for exploring protein interactions

A method is described for the elucidation of protein-protein interactions using novel cross-linking reagents and mass spectrometry. The method incorporates (1) a modular solid-phase synthetic strategy for generating the cross-linking reagents, (2) enrichment and digestion of cross-linked proteins using microconcentrators, (3) mass spectrometric analysis of cross-linked peptides, and (4) comprehensive computational analysis of the cross-linking data. This integrated approach has been applied to the study of cross-linking between the components of the heterodimeric protein complex negative cofactor 2.     

Strategy for selective chemical cross-linking of tyrosine and lysine residues

Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin beta-chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminus-tyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH.     

PERVAPORATION PROPERTIES OF PDMS MEMBRANES CURED WITH DIFFERENT CROSS-LINKING REAGENTS FOR ETHANOL CONCENTRATION FROM AQUEOUS SOLUTIONS

Ethanol perm-selective PDMS/PVDF composite membranes were prepared by curing polydimethylsiloxane (PDMS) with various cross-linking reagents,such as tetraethoxylsilane(TEOS),γ-aminopropyltriethoxylsilane(APTEOS), phenyltrimethoxylsilane(PTMOS) and octyltrimethoxylsilane(OTMOS) as well.The cross-linking density and surface properties of the PDMS active layer were adjusted by varying cross-linking reagents.The pervaporation performance of PDMS membranes cured with different cross-linking reagents was inves...     

Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes

Chemical cross-linking of proteins, an established method in protein chemistry, has gained renewed interest in combination with mass spectrometric analysis of the reaction products for elucidating low-resolution three-dimensional protein structures and interacting sequences in protein complexes. The identification of the large number of cross-linking sites from the complex mixtures generated by chemical cross-linking, however, remains a challenging task. This review describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the strategies employing intra- or intermolecular chemical cross-linking and mass spectrometry. The various approaches described in the literature to facilitate detection of cross-linking products and also computer software for data analysis are reviewed. Cross-linking techniques combined with mass spectrometry and bioinformatic methods have the potential to provide the basis for an efficient structural characterization of proteins and protein complexes.     

Cross-linking the protein precursor of marine mussel adhesives: bulk measurements and reagents for curing

Marine mussels affix themselves to surfaces by use of a highly cross-linked, protein-based adhesive. Metal levels (e.g., Fe, Zn, Cu, Mn) of the cured glue are significantly concentrated relative to surrounding waters. Specific details on the reagents used by mussels to induce protein cross-linking are not known at this time. To provide insight on the cross-linking agents and reactions taking place while curing mussel glues, we performed a study in which various compounds were tested for the ability to bring about protein curing. A precursor to adhesion, with proteins containing the unusual amino acid 3,4-dihydroxyphenylalanine, was extracted from mussel feet. Potential cross-linking agents were mixed with this gelatinous pellet. The compressibility and shear properties of the resulting material were investigated by use of a penetration test. The reagents examined included simple metal ions (e.g., Na+, Zn2+), oxidizing transition metals (e.g., Fe3+, Cr2O7(2-)), nonmetallic oxidants (e.g., H2O2,IO4-), and oxidizing enzymes (e.g., tyrosinase). We found that protein curing was brought about by simple oxidants and transition metal ions. The results show that optimal curing occurs when the reagent is an oxidizing metal ion (e.g., MnO4-, Fe3+). We conclude that marine mussels are likely to employ Mn3+ and Fe3+ for protein cross-linking and adhesive synthesis.     

Specificity of metal ion cross-linking in marine mussel adhesives

In an effort to understand the formation of marine bioadhesives, mussel protein extracts were cured with various reagents and the enhanced cross-linking ability of Fe3+ was found.     

Intra- and Inter-Molecular Cross-Linking of Peptide Ions in the Gas Phase: Reagents and Conditions

Intra-molecular and inter-molecular cross-linking of protonated polypeptide ions in the gas phase via ion/ion reactions have been demonstrated using N-hydroxysulfosuccinimide (sulfo-NHS)- based reagent anions. The initial step in the ion/ion reaction involves the formation of a long-lived complex between the peptide and reagent, which is a prerequisite for the covalent bioconjugation chemistry. The sulfonate groups on the NHS rings of the homo-bifunctional cross-linking reagents have high affinity for the protonated sites in the peptide and, therefore, facilitate the long-lived complex formation. In addition to the formation of a long-lived chemical complex, intra-molecular cross-linking also requires two unprotonated primary amine sites within a molecule where the covalent modification takes place. Alternatively, inter-molecular cross-linking demands the availability of one neutral primary amine site in each of the two peptides that are being cross-linked. Nucleophilic displacement of two sulfo-NHS groups by the amine functionalities in the peptide is a signature of the covalent cross-linking chemistry in the gas phase. Upon removal of the two sulfo-NHS groups, two amide bonds are formed between an unprotonated, primary amine group of a lysine side chain in the peptide and the carboxyl group in the reagent.     

Polymer-supported distannanes: a new and efficient synthesis of highly effective reagents for iodine atom transfer cyclisations

Polymeric distannanes were synthesised by the cross-linking of polystyrene bound tin hydride functionalities using a palladium mediated dehydrogenative coupling. The polymers were characterised by spectroscopic methods (IR and ¹³C, ¹¹⁹Sn NMR) and elemental analysis and were successfully applied to two iodine atom transfer cyclisations, performing as well as solution based hexaalkylditin reagents and significantly better than previously reported polymer-supported ditin reagents.     

Zwitterionic reagents for labeling, cross-linking and improving the performance of chemiluminescent immunoassays

Improving reagent performance in immunoassays both to enhance assay sensitivity and to minimize interference are ongoing challenges in clinical diagnostics. We describe herein the syntheses of a new class of hydrophilic reagents containing sulfobetaine zwitterions and their applications. These zwitterionic reagents are potentially useful for improving the properties of immunoassay reagents. We demonstrate for the first time that zwitterion labeling is a general and viable strategy for reducing the non-specific binding of proteins to microparticles and, to improve the aqueous solubility of hydrophobic peptides. We also describe the synthesis of zwitterionic cross-linking reagents and demonstrate their utility for peptide conjugation. In automated, chemiluminescent immunoassays, improved assay performance was observed for a hydrophobic, small analyte (theophylline) using an acridinium ester conjugate with a zwitterionic sulfobetaine linker compared to a hexa(ethylene)glycol linker. Sandwich assay performance for a large analyte (thyroid stimulating hormone) was similar for the two acridinium ester labels. These results indicate that zwitterions are complementary to poly(ethylene)glycol in improving the aqueous solubility and reducing the non-specific binding of chemiluminescent acridinium ester conjugates.     

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA–protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2′-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA–protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA–protein interactions because of high selectivity of cysteine trapping.     

Two-dimensional polymeric nanomaterials through cross-linking of polybutadiene-b-poly(ethylene oxide) monolayers at the air/water interface

Two-dimensional polymeric nanomaterials consisting of a continuously cross-linked polybutadiene (PB) two-dimensional network with poly(ethylene oxide) (PEO) domains of controlled sizes trapped within the PB network were synthesized. To reach that goal, novel (PB(Si(OEt)3)-b-PEO)3 star block copolymers were designed by hydrosilylation of the pendant double bonds of (PB-b-PEO)3 star block copolymer precursors with triethoxysilane. The (PB(Si(OEt)3)-b-PEO)3 star block copolymers were characterized by 1H NMR and IR spectroscopy. Self-condensation of the triethoxysilane pendant groups under acidic conditions led to a successful cross-linking of the polybutadiene blocks directly at the air/water interface without any additives or reagents. This strategy was found more efficient than radical cross-linking of (PB-b-PEO)3 with AIBN to get a homogeneously cross-linked monolayer of controlled and fixed morphology as demonstrated by the easy mechanical removal of the cross-linked Langmuir film from the water surface. As shown by AFM imaging, this strategy allows the accurate control of the PEO "pore" size depending on the monolayer surface pressure applied during the cross-linking reaction. The subphase pH and surface pressure influence on the cross-linking kinetics and monolayer morphologies were investigated by Langmuir trough studies (isotherm and isobar experiments) and AFM imaging.     

Mapping low-resolution three-dimensional protein structures using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry

Techniques in mass spectrometry (MS) combined with chemical cross-linking have proven to be efficient tools for the rapid determination of low-resolution three-dimensional (3-D) structures of proteins. The general procedure involves chemical cross-linking of a protein followed by enzymatic digestion and MS analysis of the resulting peptide mixture. These experiments are generally fast and do not require large quantities of protein. However, the large number of peptide species created from the digestion of cross-linked proteins makes it difficult to identify relevant intermolecular cross-linked peptides from MS data. We present a method for mapping low-resolution 3-D protein structures by combining chemical cross-linking with high-resolution FTICR (Fourier transform ion-cyclotron resonance) mass spectrometry using cytochrome c and hen egg lysozyme as model proteins. We applied several homo-bifunctional, amine-reactive cross-linking reagents that bridge distances from 6 to 16 A. The non-digested cross-linking reaction mixtures were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to determine the extent of cross-linking. Enzymatically digested reaction mixtures were separated by nano-high-performance liquid chromatography (nano-HPLC) on reverse-phase columns applying water/acetonitrile gradients with flow rates of 200 nL/min. The nano-HPLC system was directly coupled to an FTICR mass spectrometer equipped with a nano-ESI (electrospray ionization) source. Cross-linking products were identified using a combination of the GPMAW software and ExPASy Proteomics tools. For correct assignment of the cross-linking products the key factor is to rely on a mass spectrometric method providing both high resolution and high mass accuracy, such as FTICRMS. By combining chemical cross-linking with FTICRMS we were able to rapidly define several intramolecular constraints for cytochrome c and lysozyme.     

A diverse set of oligomeric class II MHC-peptide complexes for probing T-cell receptor interactions

BACKGROUND: T-cells are activated by engagement of their clonotypic cell surface receptors with peptide complexes of major histocompatibility complex (MHC) proteins, in a poorly understood process that involves receptor clustering on the membrane surface. Few tools are available to study the molecular mechanisms responsible for initiation of activation processes in T-cells. RESULTS: A topologically diverse set of oligomers of the human MHC protein HLA-DR1, varying in size from dimers to tetramers, was produced by varying the location of an introduced cysteine residue and the number and spacing of sulfhydryl-reactive groups carried on novel and commercially available cross-linking reagents. Fluorescent probes incorporated into the cross-linking reagents facilitated measurement of oligomer binding to the T-cell surface. Oligomeric MHC-peptide complexes, including a variety of MHC dimers, trimers and tetramers, bound to T-cells and initiated T-cell activation processes in an antigen-specific manner. CONCLUSION: T-cell receptor dimerization on the cell surface is sufficient to initiate intracellular signaling processes, as a variety of MHC-peptide dimers differing in intramolecular spacing and orientation were each able to trigger early T-cell activation events. The relative binding affinities within a homologous series of MHC-peptide oligomers suggest that T-cell receptors may rearrange in the plane of the membrane concurrent with oligomer binding.     

New photoactivators for multiphoton excited three-dimensional submicron cross-linking of proteins: bovine serum albumin and type 1 collagen

We report the synthesis and optical characterization of two new photoactivators and demonstrate their use for multiphoton excited three-dimensional free-form fabrication with proteins. These reagents were developed with the goal of cross-linking Type 1 collagen. This cross-linking process produces structures on the micron and submicron size scales. A rose bengal diisopropyl amine derivative combines the classic photoactivator and co-initiator system into one molecule, reducing the reaction kinetics and increasing cross-linking efficiency. This derivative was successful at producing stable structures from collagen, whereas rose bengal alone was not effective. A benzophenone dimer connected by a flexible diamine tether was also synthesized. This activator has two photochemically reactive groups and is highly efficient in cross-linking bovine serum albumin and Type 1 collagen to form stable, robust structures. This approach is more flexible in terms of cross-linking a variety of proteins than by traditional benzophenone photochemistry. The photophysical properties vary greatly from that of benzophenone, with the appearance of a new, lower energy absorption band (lambda max approximately 370 nm in water) and broad, visible emission band (approximately 500 nm maximum). This absorption band is highly solvatochromic, suggesting it arises, at least in part, from a charge transfer interaction. Collagens are typically difficult to cross-link photochemically, and the results here suggest that these two new activators will be suitable for cross-linking other forms of collagen and additional proteins for biomedical applications such as the de novo assembly of biomimetic tissue scaffolds.     

Bifunctional Reagents for Bioorganic Syntheses. Bis-Enamines. Cross-Linking by Amine Exchange Reactions

Synthesis of several 3,3′-sulfonyl-bis-enamines (7–11) is reported. These bis-enamines undergo facile amine exchange reactions with primary amines to form new, more stable bis-enamines (12–16). Bis-enamines represent a new class of bifunctional reagents with potential utility in biomacromolecular cross-linking.     

Shell cross-linked polymer micelles: stabilized assemblies with great versatility and potential

Shell cross-linked polymer micelles have been introduced within the past 3 years, and they have already demonstrated great promise as robust nanostructured core-shell nanospheres. The formation of cross-links throughout the shell of polymer micelles offers stability to the nanostructured assemblies, by providing reinforcement to the weak interactions that facilitate polymer micelle existence. Cross-linking can be accomplished by direct reaction between the chain segments located within the polymer micelle shell, or via addition of multi-functional cross-linking reagents. The dimensions, composition, and properties of each of the domains of the polymer micelles can be controlled by selection of diblock copolymer composition, conditions for polymer micelle assembly, and chemistry used for cross-linking. An overview of each of the examples of SCK nanospheres currently known is presented here.     

Purification of the isoflavonoid puerarin by adsorption chromatography on cross-linked 12% agarose

The isoflavonoid puerarin in extracts of the well-known traditional Chinese drug Radix puerariae (root of the plant Pueraria lobata) can be separated from other isoflavonoids by adsorption chromatography on the cross-linked 12% agarose gel Superose 12 equilibrated in distilled water. The adsorption is totally quenched by the addition of 50% acetic acid. The separation of the isoflavonoids is tentatively ascribed to interaction with the residues of the cross-linking reagents used in the manufacturing process of Superose 12. Thus, no useful separation can be achieved with non-cross-linked 12% agarose gel media. Symmetric elution profiles at high sample loadings (16 mg on a 24 ml column) suggest linear adsorption isotherms for the isoflavonoids.     

Optimization of cross-linking parameters during production of transglutaminase-hardened spherical multinuclear microcapsules by complex coacervation

Gelatin-gum arabic spherical multinuclear microcapsules (SMMs) encapsulating peppermint oil were prepared by complex coacervation. Transglutaminase (TG) was used to harden the SMMs by complex coacervation instead of traditional reagents such as formaldehyde or glutaraldehyde. The effect of various cross-linking parameters on the hardening effectiveness of SMMs containing peppermint oil was investigated. The optimum parameters were as follows: hardening for 6h at 15 degrees C and pH 6.0 with a TG concentration of 15 U/g gelatin. Compared with formaldehyde, TG exhibits similar microcapsule hardening effectiveness.     

AZIDOTETRAFLUOROPHENYL RETINAL ANALOGUE: SYNTHESIS AND BACTERIORHODOPSIN PIGMENT FORMATION

Abstract —The retinal derivative, all-truns-9–(4-azido-2,3,5,6-tetrafluorophenyl)-3,7-dimethyl-2,4,6,8-nonatetraenal, was synthesized by two routes as a potential photoactivatable cross-linking agent for studies in bacteriorhodopsin (BR) of the chromophore interaction with its apoprotein. The retinal analogue formed a stable, moderately functional BR pigment confirming that the ring cavity of the retinal binding site has a significant tolerance for derivatization on that portion of the molecule. Attempts to cross-link the azido chromophore to the protein by photoactivation were unsuccessful. The electron delocalization effect of the conjugated polyene side chain of the retinal appears to interfere with the formation or reactivity of the nitrene intermediate to the extent that photoactivated cross-linking is not achieved. These results demonstrate a limitation to the use of fluorinated aryl azides as photoaffinity reagents.     

Methods of obtaining lignin-carbohydrate compounds from chemically modified plant raw materials

Chemical modification of plant raw materials aimed at obtaining new valuable products and materials is described. Methods of preliminary treatment and modification of biopolymers (cavitation, microwave irradiation, and application of cross-linking and delignification reagents) making it possible to reduce decomposition of all major components are considered. Modified products can be used as sorbents of heavy metal salts, additives to drilling fluids in drilling of oil and gas wells, in the construction industry etc.