Complex Of Protein Research Articles

High-Throughput Plasma Proteomic Profiling.

Plasma and serum are rich sources of proteins that are commonly used for clinical proteome profiling and biomarkers discovery. However, high-throughput plasma proteome profiling and quantitative analysis using mass spectrometry are challenging because of the large dynamic range of protein abundance and complexity. To overcome these challenges, we developed a convenient high-throughput workflow of depleted plasma using the 4D-Proteomics feature of the Bruker timsTOF Pro mass spectrometer with data-dependent (PASEF) and data-independent acquisition (diaPASEF) method that can potentially be used in a clinical proteome profiling and biomarker discoveries. This workflow is robust, optimal for high throughput, high proteome depth, and is reproducible. In our sample preparation steps, we used immuno-depletion steps to remove high-abundance plasma proteins, and without any further cleanup steps, we can use depleted plasma samples directly for enzymatic digestion. Immuno-depletion steps and 4D-Proteomics features of timsTOF Pro increase the plasma proteome depth, and accuracy with the identification of >800 protein groups.

Effects of soybean oil and whey protein complexation by temperature treatments on the structural, physicochemical and digestive characteristics of autoclaved rice

To further reduce the digestibility of autoclaved rice, autoclaved rice flour (ARF) was added to soybean oil (SO) and whey protein (WP) and treated at different temperatures. The results showed that the treatment temperatures had a profound effect on the morphologies of the ARF; the addition of SO and WP had a protective effect on the morphologies of the ARF particles when they were subjected to thermal treatment. With increasing complexation temperature, the relative crystallinities of both the ARF and ARF-SO-WP displayed a pattern of decreasing first and then increasing. The hydrolysis rates of the ARF increased gradually with the increase of the treatment temperatures, while the hydrolysis rates of the ARF-SO-WP decreased with the increase of the treatment temperatures first, then the hydrolysis rate increased when the temperature was further increased to 70 °C. Compared with the HI and EGI of the ARF, those of the ARF-SO-WP became much lower. We could conclude that protein and lipid addition could lower the EGI of autoclaved rice.

An electrochemical paper-based analytical sensor for one-step latex protein detection.

Exposure to natural rubber latex (NRL) can result in sensitivity to NRL protein with resulting allergic reactions. Low-cost, portable, simple, sensitive analytical tools for NRL protein measurements are needed for rapid and accurate assessments of allergenic risks at the point-of-care (POC) instead of using traditional methods that require large and expensive instruments, long-time analysis, and complex sample preparation steps. Here, an electrochemical paper-based analytical device (ePAD) is presented by combining sample preparation and electrochemical detection within a single device to offer a one-step NRL analysis. The lack of antibodies and/or enzymes against NRL makes POC analysis difficult. In this work, detection is based on electrochemical measurement of the remaining Cu after in situ protein complexation instead of more complex biological assays for the first time. Graphene screen-printed electrodes modified with 1,10-phenanthroline and Nafion were used in the ePAD to improve Cu signal 18-fold relative to unmodified carbon screen-printed electrodes. The optimum parameters including 1,10-phenanthroline concentration, reaction time between Cu and protein, and the starting Cu concentration were 5% w/v, 1 min, and 600 μg mL-1, respectively. In addition to short analysis time (4 min), the system selectivity indicated no other interfering species affecting protein detection. The proposed ePAD achieved an LOD of 3.0 mg dL-1 and a linear range of 10.0-200.0 mg dL-1. Finally, the proposed sensors were applied for NRL protein detection and the results were not significantly different from the traditional Lowry method at 95% confidence.

Effect of protein complexation by pectin and small molecular surfactants

Effect of protein complexation by pectin and small molecular surfactants

Members of the vertebrate contactin and amyloid precursor protein families interact through a conserved interface

Contactins (CNTNs) are neural cell adhesion molecules that encode axon-target specificity during the patterning of the vertebrate visual and olfactory systems. Because CNTNs are tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, they lack an intracellular region to communicate across the membrane. Instead, they form coreceptor complexes with distinct transmembrane proteins to transmit signals inside the cell. In particular, a complex of CNTN4 and amyloid precursor protein (APP) is known to guide the assembly of specific circuits in the visual system. Here, using in situ hybridization in zebrafish embryos, we show that CNTN4, CNTN5, and the APP homologs, amyloid beta precursor like protein 1 and amyloid beta precursor like protein 2, are expressed in olfactory pits, suggesting that these receptors may also function together in the organization of olfactory tissues. Furthermore, we use biochemical and structural approaches to characterize interactions between members of these two receptor families. In particular, APP and amyloid beta precursor like protein 1 interact with CNTN3–5, whereas amyloid beta precursor like protein 2 only binds to CNTN4 and CNTN5. Finally, structural analyses of five CNTN–amyloid pairs indicate that these proteins interact through a conserved interface involving the second fibronectin type III repeat of CNTNs and the copper-binding domain of amyloid proteins. Overall, this work sets the stage for analyzing CNTN–amyloid-mediated connectivity in vertebrate sensory circuits.

Laccase-catalyzed soy protein and gallic acid complexation: Effects on conformational structures and antioxidant activity

The ability of laccase to oxidize polyphenols arouses our interest that laccase can be applied for protein-polyphenol cross-linking. In this study, laccase promoted the cross-linking of gallic acid (GA) and soy protein isolate (SPI) under neutral pH. SPI-GA complexes changed the secondary structures with a decrease in β-fold and an increase in α-helix and β-turn. The free-radical scavenging activity and reducing power determination results suggested that GA elevated the SPI antioxidant activity significantly. Specifically, DPPH free radical scavenging rate and ABTS free radical scavenging ability increased almost 5- and 1.5-fold compared with unmodified SPI, respectively. Moreover, the reducing power had more than 3-fold compared to the SPI control. This study provided a novel enzyme-induced approach to modulate the physicochemical properties of SPI binding polyphenol.

Making Protein-Based Adhesives Water Resistant: Role of Protein Water Solubility, Galloyl Modification, and Complexation

There has been an ongoing interest in using proteins as more environmentally friendly substitutes for the existing synthetic adhesives. Poor water resistance is a particular shortcoming of protein-based adhesives, which negatively affects their outdoor functionality. In this study, we systematically investigated if water solubility of the protein, addition of a phenolic compound such as tannic acid, and protein–polyphenol complexation could improve the water resistance of protein-based adhesives. It was found that the synergetic effects of these parameters can yield commercial-level water resistance.

Chemical-Biology-derived in vivo Sensors: Past, Present, and Future.

To understand the complex biochemistry and biophysics of biological systems, one needs to be able to monitor local concentrations of molecules, physical properties of macromolecular assemblies and activation status of signaling pathways, in real time, within single cells, and at high spatio-temporal resolution. Here we look at the tools that have been / are being / need to be provided by chemical biology to address these challenges. In particular, we highlight the utility of molecular probes that help to better measure mechanical forces and flux through key signalling pathways. Chemical biology can be used to both build biosensors to visualize, but also actuators to perturb biological processes. An emergent theme is the possibility to multiplex measurements of multiple cellular processes. Advances in microscopy automation now allow us to acquire datasets for 1000's of cells. This produces high dimensional datasets that require computer vision approaches that automate image analysis. The high dimensionality of these datasets are often not immediately accessible to human intuition, and, similarly to 'omics technologies, require statistical approaches for their exploitation. The field of biosensor imaging is therefore experiencing a multidisciplinary transition that will enable it to realize its full potential as a tool to provide a deeper appreciation of cell physiology.

Effect of protein topology on hierarchical complexation of epsilon-polylysine and protein: A multiscale structural analysis

ε-polylysine (EPL) is an antimicrobial peptide. Despite the utilization of EPL as a natural preservative worldwide, it will be deactivated owing to phase separation when it encounters some macroions. Interestingly, previous studies demonstrated the approach to preserve its antimicrobial efficacy by forming another delicate structure, the “soluble” complexes, but their interaction mechanism and structure remain unclear. This work elucidated the hierarchically structural complexation of EPL with three representative proteins β-casein, β-lactoglobulin and bovine serum albumin. It was found that these hierarchical structures separated into compact mesophase dispersions and porous precipitations, and range from nanometers to microns length scales. The driving forces for the association between EPL and protein have been mainly considered as electrostatic and subsequently hydrophobic attractions. Our results illustrated how the differences in topology (spatial structure, charge anisotropy, and hydrophobicity) among various proteins determined the specifically hierarchical structures of EPL-protein complexation. Unfolded protein with the concentrated negative charge and hydrophobicity patches inclined to form aggregation readily, whereas the folded protein with a relative disperses geometry could create the structural forces to form partial steady-state complexes. This work provides a general structural evolution of EPL-protein complexation and paved the way for its future application in food science and biotechnology.

Protein domain-based prediction of drug/compound-target interactions and experimental validation on LIM kinases.

Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network-based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins' structure/function, and bias in system training datasets. Here, we propose a new method "DRUIDom" (DRUg Interacting Domain prediction) to identify bio-interactions between drug candidate compounds and targets by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying their interactions. As such, other proteins containing the same mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including those mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting drug/compound-target pairs (~2.9M data points), and used as training data for calculating parameters of compound-domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of ~5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. Datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

Bioinspired translation of classical music into de novo protein structures using deep learning and molecular modeling

Architected biomaterials, as well as sound and music, are constructed from small building blocks that are assembled across time- and length-scales. Here we present a novel deep learning-enabled integrated algorithmic workflow to merge the two concepts for radical discovery of de novo protein materials, exploiting musical creativity as the foundation, and extrapolating through a recursive method to increase protein complexity by successively injecting protein chemistry into the process. Indeed, music is one of the few universal expressions that can create bridges between cultures, find associations between seemingly unrelated concepts, and can be used as a novel way to generate bio-inspired designs that derive functions from the imaginations of the creative mind. Earlier work has offered a pathway to convert proteins into sound, and sound into proteins. Here we build on this paradigm and translate a piece of classical music into matter. Based on Bach’s Goldberg variations, we offer a series of case studies to convert the musical data imagined by the composer into protein design, and folded into a 3D structure using deep learning. The quest we seek to address is to identify semblances, or memories, or information content in such musical creation, that offers new insights into pattern relationships between distinct manifestations of information. Using basic local alignment search tool analysis, we find that several fragments of the new proteins display similarities to existing protein sequences found in proteobacteria among other organisms, especially in regions of low complexity and repetitive motifs. The resulting protein forms the basis for iterative musical composition, and an evolutionary paradigm that defines a variational pathway for melodic development, complementing conventional creative or mathematical methods. This paper broadens the concept of what is understood as bio-inspiration to include a broad array of systems created by humans, animals, or other natural mechanisms.

Enzymes: a very short introduction by Paul Engel

This book comes from a series of ‘very short introductions’ aimed at introducing the reader to a new subject. To describe this book as a journey from simple concepts to complex biochemistry would be accurate; however, lay readers may be surprised to find themselves in high-speed pursuit and it is perhaps more appropriate for those with a scientific background. The book starts off in chapter 1 with lay descriptions of enzymes and why they are useful, raising questions such as ‘What is life?’ Chapter 2 then picks up the pace with descriptions of kinetics and the concepts associated with catalysis; however, this is restricted to an explanation of the basics of the Michaelis–Menten equation. Those seeking a more detailed exploration of enzyme kinetics would be better to target a book specific to enzyme kinetics. Subsequent chapters investigate examples of enzymes, their use in medicine and industrial applications. There is perhaps a slight bias to genetic examples; however, this is perhaps a reflection of the biological basis of the book as opposed to a chemical one. This book would be an excellent accompaniment to an undergraduate biology student or someone with a scientific background who wishes to know more about enzymology. For general scientific readers this book offers a level of detail to satisfy most questions around enzymes whilst also giving relatable examples to everyday life.

Mechanism of the structural interaction between whey and lentil proteins in the unique creation of a protein structure.

Poor solubility is a substantial factor that restricts the production of high value-added lentil proteins (LPs). In this study, whey protein isolates (WPIs), which have high solubility and are used in various food industries, were mixed with LPs at pH 12 to create LP-WPI protein complexes with improved water solubility properties using pH-recycling approach (maintained at pH 12.0 for 60min and then readjusting to pH 7.0). LP-WPI protein complexes produced in this study have gained high surface charge, increased in the solubilization of protein complexes to ≈92%, as well as improved resistance against protein aggregation. The ratio of LPs to WPIs has a significant effect on the generation of unique tertiary and secondary protein structures based on the protein-protein interaction (PPI) technique via pH-recycling. The protein interaction between LPs and WPIs resulted in alteration on the surface morphology of the produced protein complexes. This study showed that electrostatic interaction, hydrophobic force, and hydrogen bond appear as major molecular forces in this PPI. The efficacy of the pH-recycling method used in this research indicates that this approach could be a robust approach to enhance the functional properties of food proteins. PRACTICAL APPLICATION: The pH-recycling technique is a proven technique for protein complexation in creating novel protein complexes with improved functional properties. Even though lentils are a rich source of plant-based protein, its utilization by food industries is restricted due to the poor water solubility of lentil proteins (LPs). However, by using complexing lentil proteins with whey protein isolates (WPIs), that is, LP-WPI protein complex, was developed. The water solubility of LP-WPI protein complex was significantly higher than LPs, up to approximately 92%. In addition, this could improve the utilization of lentil seeds in food application as an alternative for animal-based proteins.

Predicting protein interaction network perturbation by alternative splicing with semi-supervised learning

Alternative splicing introduces an additional layer of protein diversity and complexity in regulating cellular functions that can be specific to the tissue and cell type, physiological state of a cell, or disease phenotype. Recent high-throughput experimental studies have illuminated the functional role of splicing events through rewiring protein-protein interactions; however, the extent to which the macromolecular interactions are affected by alternative splicing has yet to be fully understood. In silico methods provide a fast and cheap alternative to interrogating functional characteristics of thousands of alternatively spliced isoforms. Here, we develop an accurate feature-based machine learning approach that predicts whether a protein-protein interaction carried out by a reference isoform is perturbed by an alternatively spliced isoform. Our method, called the alternatively spliced interactions prediction (ALT-IN) tool, is compared with the state-of-the-art PPI prediction tools and shows superior performance, achieving 0.92 in precision and recall values.

Influence of calcium chloride and pH on soluble complex of whey protein-basil seed gum and xanthan gum.

SummaryInteraction between biopolymers generates different rheological behaviors, which can be effective on the structure of food products. One way to control the polysaccharide–protein interaction is the variation of acidic and ionic strength. In this research, the different amounts of pHs (3–7) and calcium chloride (5–20 mM) were investigated on a soluble complex of whey protein concentrate (WPC) with xanthan gum (XG) and basil seed gum (BSG). The complex characteristic was investigated according to turbidity, viscosity behavior, and electrostatic interactions. The turbidity test showed that WPC:BSG and WPC:XG absorbance increased at pH 3.5 and 4.5, respectively, due to the formation of insoluble complex. pH 6 was the start point of the turbidity increment, which showed the formation of soluble complexes between WPC and polysaccharides. The FTIR analysis confirmed creation of soluble complex at pH 6. The absorbance raised with increasing the molar of CaCl2 to 10 mM, but no significant difference was observed by turbidity test in the range of CaCl2<10 mM. Also, the highest viscosity value was obtained by 10 mM CaCl2.

Nano Milk Protein-Mucilage Complexes: Characterization and Anticancer Effect.

The anticancer activity of natural compounds has recently attracted multidisciplinary research. In this study, the complexation of milk proteins (MP) with Isabgol husk mucilage (IHM) and Ziziphus spina-christi mucilage (NabM) was investigated. In this context, the physicochemical properties of milk protein mucilage complexes (MPMC) including pH, Carr’s index, water solubility, and water absorption indices were measured, and the flow behavior was studied. In addition, the amino acid profile, protein digestibility, and phenolic and flavonoids content of MPMC were explored, and the microstructure of the complexes was visualized using transmission electron microscopy. The antioxidant and anticancer potencies of MPMC against two cancerous cell lines, human liver cancer HEPG-2 and breast cancer MCF-7, in comparison with two normal cell lines, namely, Bj-1 and MCF-12F, were tested using neutral red uptake assay. The results revealed that MPMC had scavenging activity against DPPH, ABTS, and HS radicals. Moreover, MPMC has the potential to prevent DNA damage induced by oxidative stress in Type-Fenton’s reaction. The results of the neutral red assay showed significant growth inhibition of both HEPG-2, MCF-7, whereas no significant cytotoxic effect was detected against Bj-1 and MCF-12F. RT-qPCR results indicated MPMC stimulated apoptosis as revealed by the upregulation of the pro-apoptosis gene markers Casepase-3, p53, Bax. Meanwhile, the anti-apoptosis Bcl-2 gene was downregulated. However, no significant difference was observed in normal cell lines treated with MPMC. In conclusion, MPMC can be considered as a promising anticancer entity that can be used in the development of novel cancer therapeutics with comparable activity and minimal side effects compared to conventional cancer chemotherapies.

Higher Order Protein Catenation Leads to an Artificial Antibody with Enhanced Affinity and In Vivo Stability.

The chemical topology is a unique dimension for protein engineering, yet the topological diversity and architectural complexity of proteins remain largely untapped. Herein, we report the biosynthesis of complex topological proteins using a rationally engineered, cross-entwining peptide heterodimer motif derived from p53dim (an entangled homodimeric mutant of the tetramerization domain of the tumor suppressor protein p53). The incorporation of an electrostatic interaction at specific sites converts the p53dim homodimer motif into a pair of heterodimer motifs with high specificity for directing chain entanglement upon folding. Its combination with split-intein-mediated ligation and/or SpyTag/SpyCatcher chemistry facilitates the programmed synthesis of protein heterocatenane or [n]catenanes in cells, leading to a general and modular approach to complex protein catenanes containing various proteins of interest. Concatenation enhances not only the target protein's affinity but also the in vivo stability as shown by its prolonged circulation time in blood. As a proof of concept, artificial antibodies have been developed by embedding a human epidermal growth factor receptor 2-specific affibody onto the [n]catenane scaffolds and shown to exhibit a higher affinity and a better pharmacokinetic profile than the wild-type affibody. These results suggest that topology engineering holds great promise in the development of therapeutic proteins.

Fragmented blind docking: a novel protein–ligand binding prediction protocol

In the present paper we propose a novel blind docking protocol based on Autodock-Vina. The developed docking protocol can provide binding site identification and binding pose prediction at the same time, by a systematical exploration of the protein volume performed with several preliminary docking calculations. In our opinion, this protocol can be successfully applied during the first steps of the virtual screening pipeline, because it provides binding site identification and binding pose prediction at the same time without visual evaluation of the binding site. After the binding pose prediction, MM/GBSA re-scoring rescoring procedures has been applied to improve the accuracy of the protein–ligand bound state. The FRAD protocol has been tested on 116 protein–ligand complexes of the Heat Shock Protein 90 – alpha, on 176 of Human Immunodeficiency virus protease 1, and on more than 100 protein–ligand system taken from the PDBbind dataset. Overall, the FRAD approach combined to MM/GBSA re-scoring can be considered as a powerful tool to increase the accuracy and efficiency with respect to other standard docking approaches when the ligand-binding site is unknown. Communicated by Ramaswamy H. Sarma

Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties.

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.

Analysis of Protein-Glycosaminoglycan Interactions Using Traveling Wave Ion-Mobility Mass Spectrometry.

Traveling wave ion-mobility mass spectrometry (TWIMS) combined with native mass spectrometry (MS) has emerged as a powerful tool for analyzing biomolecules, including complexes of protein and heparan sulfate (HS). This technique allows determination of the stoichiometry of the protein-HS interaction and information on the overall 3D molecular envelope.