Combination Of Chemical Cross-linking Research Articles

Antimicrobial peptide-modified AIE visual composite wound dressing for promoting rapid healing of infected wounds.

Wound infection is a major problem faced during wound healing. Therefore, it is necessary to develop wound dressings with excellent antimicrobial properties. Here, a smart response system of PVA-TPE/HA-AMP/SF/ALG wound dressing was prepared by a combination of chemical cross-linking and freeze-drying methods. We grafted AMP onto HA to endow the wound dressing with bacterial resistance and slow release of AMP. At the same time, the system detects bacterial activity in real time for precise antimicrobial activity (through the use of PVA-TPE) and modulates inflammation to reduce bacterial infection (through the use of AMP). In addition, the PVA-TPE/HA-AMP/SF/ALG wound dressing has a good three-dimensional mesh structure, which promotes cell proliferation, enhances collagen deposition and angiogenesis, and thus effectively promotes rapid healing of infected wounds. Moreover, it can induce the expression of inflammatory factors such as VEGF, TNF-α, IFN-γ, IL-4 and TGF-β1 in infected wounds through the Wnt/CAMK/p-PKC signaling pathway, inhibit inflammatory responses, promote wound healing and reduce scar formation. Therefore, the PVA-TPE/HA-AMP/SF/ALG wound dressing smart response system shows great promise in infected wound healing.

Hyaluronic Acid Hydrogels for Controlled Pulmonary Drug Delivery-A Particle Engineering Approach.

Hydrogels warrant attention as a potential material for use in sustained pulmonary drug delivery due to their swelling and mucoadhesive features. Herein, hyaluronic acid (HA) is considered a promising material due to its therapeutic potential, the effect on lung inflammation, and possible utility as an excipient or drug carrier. In this study, the feasibility of using HA hydrogels (without a model drug) to engineer inhalation powders for controlled pulmonary drug delivery was assessed. A combination of chemical crosslinking and spray-drying was proposed as a novel methodology for the preparation of inhalation powders. Different crosslinkers (urea; UR and glutaraldehyde; GA) were exploited in the hydrogel formulation and the obtained powders were subjected to extensive characterization. Compositional analysis of the powders indicated a crosslinked structure of the hydrogels with sufficient thermal stability to withstand spray drying. The obtained microparticles presented a spherical shape with mean diameter particle sizes from 2.3 ± 1.1 to 3.2 ± 2.9 μm. Microparticles formed from HA crosslinked with GA exhibited a reasonable aerosolization performance (fine particle fraction estimated as 28 ± 2%), whereas lower values were obtained for the UR-based formulation. Likewise, swelling and stability in water were larger for GA than for UR, for which the results were very similar to those obtained for native (not crosslinked) HA. In conclusion, microparticles could successfully be produced from crosslinked HA, and the ones crosslinked by GA exhibited superior performance in terms of aerosolization and swelling.

The spatial network of skeletal proteins in a stony coral.

Coral skeletons are materials composed of inorganic aragonitic fibres and organic molecules including proteins, sugars and lipids that are highly organized to form a solid biomaterial upon which the animals live. The skeleton contains tens of proteins, all of which are encoded in the animal genome and secreted during the biomineralization process. While recent advances are revealing the functions and evolutionary history of some of these proteins, how they are spatially arranged in the skeleton is unknown. Using a combination of chemical cross-linking and high-resolution tandem mass spectrometry, we identify, for the first time, the spatial interactions of the proteins embedded within the skeleton of the stony coral Stylophora pistillata. Our subsequent network analysis revealed that several coral acid-rich proteins are invariably associated with carbonic anhydrase(s), alpha-collagen, cadherins and other calcium-binding proteins. These spatial arrangements clearly show that protein–protein interactions in coral skeletons are highly coordinated and are key to understanding the formation and persistence of coral skeletons through time.

Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule–induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.

Highly compressible and durable superhydrophobic cellulose aerogels for oil/water emulsion separation with high flux

Nowadays high porosity aerogels are widely applied in wastewater purification, while the fragility and unversatility of nascent cellulose aerogels limited the application in oil/water filtering separation. Here, highly compressible, elastic, superhydrophobic cellulose/poly(vinyl alcohol) (PVA) composite aerogels were fabricated via combination of chemical cross-linking, freeze drying and silanization. The obtained cellulose/PVA aerogels exhibited durable superhydrophobicity with WCA of 156.6 °, even maintaining 155.0 ° after 30 days. Synergistic effects of physical entanglement, chemical cross-linking and H-bond network between cellulose and PVA molecular chains endowed the aerogels with excellent compressibility and recoverability. The aerogels exhibited maximum compressive stress of 490.7 kPa at 90% strain, even without reduction in mechanical strength under 70% compressive strain for 10 cycles. More importantly, the strengthened aerogels exhibited remarkable separation performances for both immiscible oil/water mixtures and surfactant-stabilized water-in-oil emulsions. Fascinating permeation flux of up to 7176.3 L m−2 h−1 and total separation amount as high as 4550.6 L m−2 with separation efficiency of 98.5% for water-in-cyclohexane emulsion were harvested. Thus, this work provides a green, sustainable and mass-producible cellulose-based aerogel for high-efficiency oily wastewater remediation.

Influence of cross-linker polarity on selectivity towards lysine side chains

The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein. SignificanceEven though the amine reactive BS2G and DSG cross-linkers have the same length of spacer and are based on N-hydroxysuccinimidic group, our data showed that each of them formed preferentially different cross-links. We demonstrated that the choice of cross-linker can have a significant impact on the output data for structural characterization of biomolecules. Using equimolar mixtures of DSG with d6-BS2G, and BS2G with d6-DSG, we established that the polar BS2G preferentially bound to polar regions of modified molecule, whereas non-polar DSG bound to hydrophobic regions. This phenomenon established that the mixture of polar and non-polar cross-linkers acted as an efficient tool for the determination of distance constraints in proteins.

Nascent Proteins Interact with Key Regions of the Outer Surface of the Ribosome

While in vitro protein folding is reasonably well understood, very little is known about how nascent chains achieve their native state in the complex cellular environment. The earliest stages of protein folding as nascent chains emerge out of the ribosome are still poorly characterized. Proteins and rRNA located within the ribosomal exit tunnel and outer surface, for instance, may interact with nascent proteins and affect their folding. Previous studies in the Cavagnero group showed that ribosome-bound nascent chains (RNCs) derived from the intrinsically disordered protein PIR and its highly negatively charged variants exhibit a significant spatial bias, consistent with the presence of non-covalent interactions between RNCs and the ribosomal surface. In this work, we probe RNC-ribosomal-protein contacts by a combination of chemical crosslinking and Western blotting, and show direct evidence for well-defined interactions between RNCs of PIR and specific ribosomal proteins. Surprisingly, the interacting ribosomal proteins are clustered only on one side of the ribosomal surface, relative to the exit tunnel. The observed interactions are particularly strong when about 15 amino acids have emerged outside of the ribosomal exit tunnel, and get weaker as the chain gets longer. This result suggests that the interactions are targeted towards the prevention of co-translational aggregation. This work corroborates an emerging view according to which the ribosome plays a fundamental role in protein biogenesis that extends well beyond the mere catalysis of peptide bond formation.

Rapid determination of quaternary protein structures in complex biological samples

The understanding of complex biological systems is still hampered by limited knowledge of biologically relevant quaternary protein structures. Here, we demonstrate quaternary structure determination in biological samples using a combination of chemical cross-linking, high-resolution mass spectrometry and high-accuracy protein structure modeling. This approach, termed targeted cross-linking mass spectrometry (TX-MS), relies on computational structural models to score sets of targeted cross-linked peptide signals acquired using a combination of mass spectrometry acquisition techniques. We demonstrate the utility of TX-MS by creating a high-resolution quaternary model of a 1.8 MDa protein complex composed of a pathogen surface protein and ten human plasma proteins. The model is based on a dense network of cross-link distance constraints obtained directly in a mixture of human plasma and live bacteria. These results demonstrate that TX-MS can increase the applicability of flexible backbone docking algorithms to large protein complexes by providing rich cross-link distance information from complex biological samples.

Novel hydroxyethyl chitosan/cellulose scaffolds with bubble-like porous structure for bone tissue engineering

Hydrogels fabricated from natural polysaccharides may serve as ideal scaffolds for tissue engineering because of their similarity to the extracellular matrices. In this study, novel hydrogel scaffolds with bubble-like porous structure were prepared from hydroxyethyl chitosan (HECS) and cellulose (CEL) by a combination of chemical crosslinking, particle-leaching using silicon dioxide particles as porogen and freeze-drying method. The morphology, compression stress-strain curves, wettability, and swelling and rheological behaviors of the HECS/CEL scaffolds were characterized by SEM, mechanical test, contact angle measurement and rheometer. HECS/CEL scaffolds had good comprehensive performances and could reach equilibrium swelling state in water within 20s. The results from in vitro biocompatibility evaluated using SEM, live/dead cell viability and MTT assays demonstrated that the HECS/CEL scaffolds could well support the attachment, spreading and proliferation of osteoblastic MC3T3-E1 cells and showed good biocompatibility. Therefore, the novel HECS/CEL scaffolds may be promising for bone tissue engineering applications.

Mapping the Ca2 + induced structural change in calreticulin

Calreticulin is a highly conserved multifunctional protein implicated in many different biological systems and has therefore been the subject of intensive research. It is primarily present in the endoplasmatic reticulum where its main functions are to regulate Ca2+ homeostasis, act as a chaperone and stabilize the MHC class I peptide-loading complex. Although several high-resolution structures of calreticulin exist, these only cover three-quarters of the entire protein leaving the extended structures unsolved. Additionally, the structure of calreticulin is influenced by the presence of Ca2+. The conformational changes induced by Ca2+ have not been determined yet as they are hard to study with traditional approaches. Here, we investigated the Ca2+-induced conformational changes with a combination of chemical cross-linking, mass spectrometry, bioinformatics analysis and modelling in Rosetta. Using a bifunctional linker, we found a large Ca2+-induced change to the cross-linking pattern in calreticulin. Our results are consistent with a high flexibility in the P-loop, a stabilization of the acidic C-terminal and a relatively close interaction of the P-loop and the acidic C-terminal. Biological significanceThe function of calreticulin, an endoplasmatic reticulin chaperone, is affected by fluctuations in Ca2+concentration, but the structural mechanism is unknown. The present work suggests that Ca2+-dependent regulation is caused by different conformations of a long proline-rich loop that changes the accessibility to the peptide/lectin-binding site. Our results indicate that the binding of Ca2+ to calreticulin may thus not only just be a question of Ca2+ storage but is likely to have an impact on the chaperone activity.

Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors.

Critical Assessment of Protein Cross-Linking and Molecular Docking: An Updated Model for the Interaction Between Photosystem II and Psb27.

Photosystem II (PSII) is a large membrane-protein complex composed of about 20 subunits and various cofactors, which mediates the light-driven oxidation of water and reduction of plastoquinone, and is part of the photosynthetic electron transfer chain that is localized in the thylakoid membrane of cyanobacteria, algae, and plants. The stepwise assembly of PSII is guided and facilitated by numerous auxiliary proteins that play specific roles in this spatiotemporal process. Psb27, a small protein localized in the thylakoid lumen, appears to associate with an intermediate PSII complex that is involved in assembly of the Mn4CaO5 cluster. Its precise binding position on the PSII intermediate remains elusive, as previous approaches to the localization of Psb27 on PSII have yielded contradictory results. This was our motivation for a critical assessment of previously used methods and the development of an improved analysis pipeline. The combination of chemical cross-linking and mass spectrometry (CX-MS) with isotope-coded cross-linkers was refined and validated with reference to the PSII crystal structure. Psb27 was localized on the PSII surface adjacent to the large lumenal domain of CP43 on the basis of a cross-link connecting Psb27-K91 to CP43-K381. Additional contacts associating Psb27 with CP47 and the C-termini of D1 and D2 were detected by surface plasmon resonance (SPR) spectroscopy. This information was used to model the binding of Psb27 to the PSII surface in a region that is occupied by PsbV in the mature complex.

Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes.

During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this "traditional" cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.

The NdhV subunit is required to stabilize the chloroplast NADH dehydrogenase-like complex in Arabidopsis.

The chloroplast NADH dehydrogenase-like (NDH) complex is involved in cyclic electron transport around photosystem I (PSI) and chlororespiration. Although the NDH complex was discovered more than 20 years ago, its low abundance and fragile nature render it recalcitrant to analysis, and it is thought that some of its subunits remain to be identified. Here, we identified the NDH subunit NdhV that readily disassociates from the NDH complex in the presence of detergent, salt and alkaline solutions. The Arabidopsis ndhv mutant is partially defective in the accumulation of NDH subcomplex A (SubA) and SubE, resulting in impaired NDH activity. NdhV was mainly detected in the wild-type thylakoid membrane, and its accumulation in thylakoids strictly depended on the presence of the NDH complex. Quantitative immunoblot analysis revealed that NdhV and NdhN occur at close to equimolar concentrations. Furthermore, several NDH subunits were co-immunopurified with NdhV using a combination of chemical crosslinking and an affinity chromatography assay. These data indicate that NdhV is an intrinsic subunit of NDH. We found that NdhV did not directly affect NDH activity, but that NDH SubA and SubE were more rapidly degraded in ndhv than in the wild type under high-light treatment. We propose that NdhV is an NDH subunit that stabilizes this complex, especially under high-light conditions.

Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.

The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.

Combinatorial Approach for Large-scale Identification of Linked Peptides from Tandem Mass Spectrometry Spectra

The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein-protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides. Here we propose a generic approach to address this problem and demonstrate it using disulfide-bridged peptide libraries to (i) efficiently generate large mass spectral reference data for linked peptides at a low cost and (ii) automatically train an algorithm that can efficiently and accurately identify linked peptides from MS/MS spectra. We show that using this approach we were able to identify thousands of MS/MS spectra from disulfide-bridged peptides through comparison with proteome-scale sequence databases and significantly improve the sensitivity of cross-linked peptide identification. This allowed us to identify 60% more direct pairwise interactions between the protein subunits in the 20S proteasome complex than existing tools on cross-linking studies of the proteasome complexes. The basic framework of this approach and the MS/MS reference dataset generated should be valuable resources for the future development of new tools for the identification of linked peptides.

Architecture of an RNA polymerase II transcription pre-initiation complex.

The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.

Dermokine-β impairs ERK signaling through direct binding to GRP78

Dermokine-β is abundant in stratified epithelia and in differentiating cultured keratinocytes. In this study, we investigated the role of dermokine-β in differentiation of keratinocytes. Treatment of keratinocytes or skin tumor cells with dermokine-β attenuated phosphorylation of extracellular-signal-regulated kinase (ERK). Exposure of cells to dermokine-β, as well as its carboxyl-terminus domain peptide, interrupted phosphorylation of ERK and stimulated dermokine gene expression. Inhibition of ERK signaling by its specific inhibitor also increased dermokine expression level. A combination of chemical cross-linking and immunoprecipitation, followed by proteomics analyses, identified glucose-regulated protein 78 (GRP78) as a dermokine-β-associated protein. Blockage of GRP78 expression by a specific siRNA abrogated actions of dermokine-β. These findings provide novel insights into the physiological significance of dermokine-β in the epidermis. Structured summary of protein interactionsdermokine-Beta physically interacts with GRP78 by cross-linking study (View interaction)dermokine-Beta(Glo2) and GRP78 physically interact by competition binding (View interaction)dermokine-Beta binds to GRP78 by anti bait coimmunoprecipitation (View interaction)

Chemical Cross-Linking and H/D Exchange for Fast Refinement of Protein Crystal Structure

A combination of chemical cross-linking and hydrogen-deuterium exchange coupled to high resolution mass spectrometry was used to describe structural differences of NKR-P1A receptor. The loop region extended from the compact core in the crystal structure was found to be closely attached to the protein core in solution. Our approach has potential to refine protein structures in solution within a few days and has very low sample consumption.

Mapping protein receptor–ligand interactions via in vivo chemical crosslinking, affinity purification, and differential mass spectrometry

Protein receptor–ligand interactions play important roles in mediating enzyme catalysis, signal transduction, and other protein functions. Immunoaffinity purification followed by mass spectrometry analysis is a common method for identifying protein receptor–ligand complexes. However, it is difficult to distinguish between specific protein binding partners and non-specifically bound proteins that co-purify with the complex. In addition, weakly interacting binding partners may dissociate from the protein receptor–ligand complexes during immunoaffinity purification. The combination of chemical crosslinking, affinity purification, and differential mass spectrometry analysis provides a direct method for capturing stable, weak, and transient protein interactions that occur in vivo and in vitro. This approach enables the identification of functional receptor–ligand binding partners with high confidence. Herein, we describe a differential mass spectrometry approach coupled with in situ chemical crosslinking and immunoaffinity purification for identifying receptor–ligand binding partners. In particular, we identified a functional, counter-ligand structure of the natural killer cell p30-related protein.