Composition Of Amino Acid Residues Research Articles

Study of bromelain and carboxymethyl cellulose surface interaction features in the process of their adsorption immobilisation

Adsorption immobilisation is a common approach to stabilise the catalytic activity of enzymes. It involves the attachment of their macromolecules to the surface of a carrier due to weak physical interactions and hydrogen bonds. According to modern concepts, this process is the ‘gentlest’ immobilisation technique in terms of the structure of the enzyme globule. Despite this fact, the activity of the immobilised preparation is often lower compared to the native enzyme. To understand the reasons, it is necessary to study the specific features of interactions within the enzyme-carrier system and to identify the types of interactions occurring between its components. When studying the structure of enzymes, it is critical to determine the nature and qualitative composition of amino acid residues on the surface of the enzyme macromolecule that interact with the carrier. If catalytically important residues are involved in the process, there may be a significant decrease in the enzyme activity. In this regard, the aim of the work was to study the features of interaction between bromelain, which is a cysteine protease, and carboxymethyl cellulose by desorption of the enzyme from the formed complex under different conditions (in the presence of ammonium sulphate or surfactant Triton X-100, including at different temperatures), as well as by flexible molecular docking. The studied substances are promising components for the production of biocatalysts for food or biomedical applications, so the study of their interaction features will expand the applications of bromelain. We determined that incubation of bromelain immobilised on carboxymethyl cellulose in solutions of ammonium sulphate with a concentration of 32 mM or higher and Triton X-100 with a concentration of 100 mM or higher resulted in the destruction of the complex and the desorption of the enzyme. It confirms that non-covalent interactions contribute to the formation of immobilised preparation. When we increased the incubation temperature of the complex above 60 °C, we also observed the release of the enzyme from the preparation, indicating the formation of hydrogen bonds between the enzyme and the carrier. In silico study confirmed the formation of these types of bonds and interactions. It was determined that the amino acid residues forming the active site of bromelain also formed bonds with the carrier.

Novel salty peptides derived from bovine bone: Identification, taste characteristic, and salt-enhancing mechanism

Excessive sodium intake is a major contributor to the incidence of cardiovascular diseases. The objective of this study was to prepare, isolate, and characterize peptides from bovine bone protein and investigate the salty/salt-enhancing mechanism of peptides. 1032 peptides were identified in the enzymatic hydrolysates of bovine bone protein and were further screened by the composition of amino acid residues and molecular docking analysis. 5 peptides were finally selected for solid-phase synthesis, and KER showed a better salty taste by sensory verification. Moreover, the synergistic effect of KER in NaCl and MSG solution could enhance the salty intensity by 65.26 %. The binding of KER to the salty receptor (TMC4) was driven by hydrogen bonding and electrostatic interactions with a binding energy of −88.0734 kcal/mol. This work may provide a new approach to efficiently screen salty peptides from natural food materials, which were expected as a taste enhancer used in salt-reducing foods.

Acetylcholinesterase Adsorption on Modified Gold: Effect of Surface Chemistry on Enzyme Binding and Activity.

Surface chemistry plays a crucial role in the performance of biosensors and biocatalysts, where enzymes directly interact with a solid support. In this work, we investigated the effect of surface charge and hydrophobicity on the binding and activity of acetylcholinesterase (AChE) following direct adsorption to modified gold surfaces. Surface modifications included self-assembled monolayers (SAMs) terminated with -COO-, -NH3+, -OH, and -CH3 functional groups at varying mole %. We also investigated the effects of positively and negatively charged helical peptides covalently coupled to the SAM. Using spectroscopic ellipsometry, we measured the surface concentration of AChE on each modified surface after 1 h of adsorption. We found that surface concentration was directly proportional to surface hydrophobicity (r = 0.76). The highest binding was observed on the more hydrophobic surfaces. We also measured the specific activity of AChE on each surface using a colorimetric assay and found that activity was inversely proportional to surface hydrophobicity (r = -0.71). The highest activity was observed on the more hydrophilic surfaces. Plotting specific activity versus surface concentration showed a similar relationship, with the highest activity observed at low AChE densities (∼20% of a monolayer) on surfaces terminated with 50% -COO- or -NH3+ and 50% -CH3 functional groups. Interestingly, this is similar to the approximate composition of hydrophobic versus hydrophilic amino acid residues on the surface of AChE. These surfaces also exhibited the highest total activity: a ∼100% improvement over bare gold due to a combination of moderate binding and high activity retention. This work highlights the importance of developing new attachment strategies beyond direct adsorption that promote, tune, and optimize both high binding and high activity retention.

Cu+/Ag+ Competition in Type I Copper Proteins (T1Cu).

Due to the similarity in the basic coordination behavior of their mono-charged cations, silver biochemistry is known to be linked to that of copper in biological systems. Still, Cu+/2+ is an essential micronutrient in many organisms, while no known biological process requires silver. In human cells, copper regulation and trafficking is strictly controlled by complex systems including many cytosolic copper chaperones, whereas some bacteria exploit the so-called "blue copper" proteins. Therefore, evaluating the controlling factors of the competition between these two metal cations is of enormous interest. By employing the tools of computational chemistry, we aim to delineate the extent to which Ag+ might be able to compete with the endogenous copper in its Type I (T1Cu) proteins, and where and if, alternatively, it is handled uniquely. The effect of the surrounding media (dielectric constant) and the type, number, and composition of amino acid residues are taken into account when modelling the reactions in the present study. The obtained results clearly indicate the susceptibility of the T1Cu proteins to a silver attack due to the favorable composition and geometry of the metal-binding centers, along with the similarity between the Ag+/Cu+-containing structures. Furthermore, by exploring intriguing questions of both metals' coordination chemistry, an important background for understanding the metabolism and biotransformation of silver in organisms is provided.

PCNA from Thermococcus gammatolerans: A protein involved in chromosomal DNA metabolism intrinsically resistant at high levels of ionizing radiation.

Proliferating cell nuclear antigen (PCNA) is an essential protein for cell viability in archaea and eukarya, since it is involved in DNA replication and repair. In order to obtain insights regarding the characteristics that confer radioresistance, the structural study of the PCNA from Thermococcus gammatolerans (PCNATg ) in a gradient of ionizing radiation by X-ray crystallography was carried out, together with a bioinformatic analysis of homotrimeric PCNA structures, their sequences, and their molecular interactions. The results obtained from the datasets and the accumulated radiation dose for the last collection from three crystals revealed moderate and localized damage, since even with the loss of resolution, the electron density map corresponding to the last collection allowed to build the whole structure. Attempting to understand this behavior, multiple sequence alignments, and structural superpositions were performed, revealing that PCNA is a protein with a poorly conserved sequence, but with a highly conserved structure. The PCNATg presented the highest percentage of charged residues, mostly negatively charged, with a proportion of glutamate more than double aspartate, lack of cysteines and tryptophan, besides a high number of salt bridges. The structural study by X-ray crystallography reveals that the PCNATg has the intrinsic ability to resist high levels of ionizing radiation, and the bioinformatic analysis suggests that molecular evolution selected a particular composition of amino acid residues, and their consequent network of synergistic interactions for extreme conditions, as a collateral effect, conferring radioresistance to a protein involved in the chromosomal DNA metabolism of a radioresistant microorganism.

Clustering of Aromatic Residues in Prion-like Domains Can Tune the Formation, State, and Organization of Biomolecular Condensates.

In immature oocytes, Balbiani bodies are conserved membraneless condensates implicated in oocyte polarization, the organization of mitochondria, and long-term organelle and RNA storage. In Xenopus laevis, Balbiani body assembly is mediated by the protein Velo1. Velo1 contains an N-terminal prion-like domain (PLD) that is essential for Balbiani body formation. PLDs have emerged as a class of intrinsically disordered regions that can undergo various different types of intracellular phase transitions and are often associated with dynamic, liquid-like condensates. Intriguingly, the Velo1 PLD forms solid-like assemblies. Here we sought to understand why Velo1 phase behavior appears to be biophysically distinct from that of other PLD-containing proteins. Through bioinformatic analysis and coarse-grained simulations, we predict that the clustering of aromatic residues and the amino acid composition of residues between aromatics can influence condensate material properties, organization, and the driving forces for assembly. To test our predictions, we redesigned the Velo1 PLD to test the impact of targeted sequence changes in vivo. We found that the Velo1 design with evenly spaced aromatic residues shows rapid internal dynamics, as probed by fluorescent recovery after photobleaching, even when recruited into Balbiani bodies. Our results suggest that Velo1 might have been selected in evolution for distinctly clustered aromatic residues to maintain the structure of Balbiani bodies in long-lived oocytes. In general, our work identifies several tunable parameters that can be used to augment the condensate material state, offering a road map for the design of synthetic condensates.

An In silico Search for the Sites of Potential Binding with Charged and Hydrophobic Carriers in the Molecules of Endoinulinase from Aspergillus ficuum and Exoinulinase from Aspergillus awamori

Abstract—The composition and location of charged and hydrophobic amino-acid residues in the endoinulinase (3SC7) and exoinulinase (1Y4W) molecules have been investigated. The percentage of different types of amino acids on the surface of enzyme molecules has been determined. It has been found that the charged and hydrophobic amino-acid residues are distributed unevenly over the surface of the molecules and form the sites of local clusters. It has been shown that positively charged carriers are the most promising for the immobilization of endoinulinases; they may interact with one of the following sites on the surface of the enzyme molecules, which are remote from the active center and contain Glu239, Glu243, Glu246, Glu247, and Asp275 (site 1); Asp399, Asp424, Glu433, Glu452, Glu453, Asp454, and Glu497 (site 2); Asp37, Asp92, Glu93, and Glu516 (site 3). Negatively charged carriers are most promising for exoinulinase, because the binding sites for these carriers that contain Lys225 and Lys247 (site 1); His80, Lys84, Lys118, and Lys121 (site 2); Lys381, Arg382, Arg387, Lys390, Lys407, Lys415, Lys417, Lys479, Arg526, and Lys531 (site 3) are located on the surface of the enzyme molecules remote from the active center. Hydrophobic carriers are the least promising, because the amino-acid residues on the surface of the enzymes, to which the polymers can bind, are in the area of the active center; these residues are Tyr39, Pro62, Val66, Leu172, Gly196, and Gly259, in the case of endoinulinase; and Tyr47, Trp65, Pro85, Val86, Leu89, Leu161, Pro232, Gly237, Pro266, Pro270, Val293, Pro298, Gly299, and Gly320, in the case of exoinulinase.

Quantitative Binding Behavior of Intrinsically Disordered Proteins to Nanoparticle Surfaces at Individual Residue Level.

The quantitative and predictive understanding how intrinsically disordered proteins (IDPs) interact with engineered nanoparticles has potentially important implications for new therapeutics as well as nanotoxicology. Based on a recently developed solution 15 N NMR relaxation approach, the interactions between four representative IDPs with silica nanoparticles are reported at atomic detail. Each IDP possesses distinct binding modes, which can be quantitatively explained by the local amino-acid residue composition using a "free residue interaction model". The model was parameterized using the binding affinities of free proteinogenic amino acids along with long-range effects, derived by site-specific mutagenesis, that exponentially scale with distance along the primary sequence. The model, which is accessible through a web server, can be applied to predict the residue-specific binding affinities of a large number of IDPs.

Free-Energy-Based Protein Design: Re-Engineering Cellular Retinoic Acid Binding Protein II Assisted by the Moveable-Type Approach.

How to fine-tune the binding free energy of a small-molecule to a receptor site by altering the amino acid residue composition is a key question in protein engineering. Indeed, the ultimate solution to this problem, to chemical accuracy (±1 kcal/mol), will result in profound and wide-ranging applications in protein design. Numerous tools have been developed to address this question using knowledge-based models to more computationally intensive molecular dynamics simulations-based free energy calculations, but while some success has been achieved there remains room for improvement in terms of overall accuracy and in the speed of the methodology. Here we report a fast, knowledge-based movable-type (MT)-based approach to estimate the absolute and relative free energy of binding as influenced by mutations in a small-molecule binding site in a protein. We retrospectively validate our approach using mutagenesis data for retinoic acid binding to the Cellular Retinoic Acid Binding Protein II (CRABPII) system and then make prospective predictions that are borne out experimentally. The overall performance of our approach is supported by its success in identifying mutants that show high or even sub-nano-molar binding affinities of retinoic acid to the CRABPII system.

Peptide retention prediction using hydrophilic interaction liquid chromatography coupled to mass spectrometry

A model that predicts retention for peptides using a HALO® penta-HILIC column and gradient elution was created. Coefficients for each amino acid were derived using linear regression analysis and these coefficients can be summed to predict the retention of peptides. This model has a high correlation between experimental and predicted retention times (0.946), which is on par with previous RP and HILIC models. External validation of the model was performed using a set of H. pylori samples on the same LC–MS system used to create the model, and the deviation from actual to predicted times was low. Apart from amino acid composition, length and location of amino acid residues on a peptide were examined and two site-specific corrections for hydrophobic residues at the N-terminus as well as hydrophobic residues one spot over from the N-terminus were created.

Pair of Stereodynamic Chiral Benzylicaldehyde Probes for Determination of Absolute Configuration of Amino Acid Residues in Peptides by Mass Spectrometry

This paper describes a simple method to determine the absolute configuration of amino acids residues in peptides by mass spectrometry using a newly developed pair of mass-tagged chiral probes without the requirement of reference standards. A pair of benzylicaldehyde probes, 1-(S)-1H in S configuration and 2-(R)-2D in deuterium-labeled R configuration with the ratio of 1:1, were synthesized for in situ condensation with amino acid residues and transformed into a pair of stereodynamic imine products. The characteristic intensity difference observed in mass spectrometry can be used to determine the absolute configuration and to quantify the enantiomeric composition of chiral amino acid residues. Significant chiral recognition ability was achieved for 18 natural chiral amino acids and for one β-amino acid by comparing the ion intensity ratio of imine products I[1-(S)-1H-AA]- to I[2-(R)-2D-AA]-. For 16 kinds of amino acids, the L form of the amino acids was more reactive with 1-(S)-1H, while D configuration amino acids preferred to react with 2-(R)-2D. However, for three kinds of amino acid, the opposite result was obtained. The configurations of the residues in the peptides, Phe-Tyr-Ala, D-Phe-Tyr-Ala, Val-Pro-Phe-D-Leu-Met, Val-Pro-Phe-Leu-D-Met, as well as in a natural peptide with unknown chirality were determined by acid hydrolysis followed by the present method. In addition, molecular modeling results illustrate that the recognition process is mainly controlled by kinetic factors. Using the new probes coupled with a mass spectrometry approach avoids time-consuming workup and separation steps. We expect that the probes could be applied as tools to determine the absolute configuration of amino acid residues in proteins in future research.

RINspector: a Cytoscape app for centrality analyses and DynaMine flexibility prediction.

MotivationProtein function is directly related to amino acid residue composition and the dynamics of these residues. Centrality analyses based on residue interaction networks permit to identify key residues in a protein that are important for its fold or function. Such central residues and their environment constitute suitable targets for mutagenesis experiments. Predicted flexibility and changes in flexibility upon mutation provide valuable additional information for the design of such experiments.ResultsWe combined centrality analyses with DynaMine flexibility predictions in a Cytoscape app called RINspector. The app performs centrality analyses and directly visualizes the results on a graph of predicted residue flexibility. In addition, the effect of mutations on local flexibility can be calculated.Availability and implementationThe app is publicly available in the Cytoscape app store.Supplementary information Supplementary data are available at Bioinformatics online.

P066 Structural aspects of HLA class I epitopes reacting with human alloantiserum

Aim Interaction between complementarity determining regions (CDR) of the antibody (Ab) and HLA (contact surface area of 700–900 A2) can be detected by mean fluorescence intensity (MFI) or lysis of cell membrane by complement dependent cytotoxicity (CDC). These biological functions of Ab depend on amino acid residue (AAR) composition of functional and structural epitopes (Ep) of the antigen. The aim of this study was to analyze Ep structure of HLA in order to interpret reactivity of Abs in the serum of kidney recipient (KR). Methods Ab analysis (IgG) and complement binding activity (C1q test) of alloantibodies were analyzed using solid phase single antigen (One Lambda, Inc.) (positive cut off MFI = 280–500) and cell based CDCx3 washes assays. Ep analysis was conducted by using HLAMatchmaker computer algorithm (MFI = 1000 was used as a positive cut off). Results Various combinations of four (63EE, 82LR, 127K, and 113YH) eplet-defined Eps were detected in HLAs Abs in KR serum reacted with, when SAA was applied. Consensus of AAR within 15 A radius of aforementioned eplets comprised sixteen positions. The results of reactivity of specific eplet carrying alleles in three assays are presented in Fig. 1. Our findings indicate that Ab reactivity is associated with AAR composition of structural Eps involved in contact with CDR of IgG. Binding to HLA likely depends on AARs at positions 44, 152, 156, and 158, whereas C1q reactivity is affected by AARs at 9H/24T and 163E/167S positions, and CDC reactivity is abolished by tryptophan (W) at 95 position. Conclusions Biological function of anti-HLA Ab requires not only the specific recognition of eplets but also depends on the interactions of their CDRs with structural Eps within the contact surface area. C1q + test does not always predict a positive CDC assay. The results of this study represent a new insight into organ allocation, immunomodulation therapy, and post-transplant Ab monitoring in solid organ recipients. Download : Download high-res image (603KB) Download : Download full-size image

Charged groups at binding interfaces of the PsbO subunit of photosystem II: A combined bioinformatics and simulation study

PsbO is an extrinsic subunit of photosystem II engaged in complex binding interactions within photosystem II. At the interface between PsbO, D1 and D2 subunits of photosystem II, a cluster of charged and polar groups of PsbO is part of an extended hydrogen-bond network thought to participate in proton transfer. The precise role of specific amino acid residues at this complex binding interface remains a key open question. Here, we address this question by carrying out extensive bioinformatics analyses and molecular dynamics simulations of PsbO proteins with mutations at the binding interface. We find that PsbO proteins from cyanobacteria vs. plants have specific preferences for the number and composition of charged amino acid residues that may ensure that PsbO proteins avoid aggregation and expose long unstructured loops for binding to photosystem II. A cluster of conserved charged groups with dynamic hydrogen bonds provides PsbO with structural plasticity at the binding interface with photosystem II.

Toxin bioportides: exploring toxin biological activity and multifunctionality.

Toxins have been shown to have many biological functions and to constitute a rich source of drugs and biotechnological tools. We focus on toxins that not only have a specific activity, but also contain residues responsible for transmembrane penetration, which can be considered bioportides-a class of cell-penetrating peptides that are also intrinsically bioactive. Bioportides are potential tools in pharmacology and biotechnology as they help deliver substances and nanoparticles to intracellular targets. Bioportides characterized so far are peptides derived from human proteins, such as cytochrome c (CYCS), calcitonin receptor (camptide), and endothelial nitric oxide synthase (nosangiotide). However, toxins are usually disregarded as potential bioportides. In this review, we discuss the inclusion of some toxins and molecules derived thereof as a new class of bioportides based on structure activity relationship, minimization, and biological activity studies. The comparative analysis of the amino acid residue composition of toxin-derived bioportides and their short molecular variants is an innovative analytical strategy which allows us to understand natural toxin multifunctionality in vivo and plan novel pharmacological and biotechnological products. Furthermore, we discuss how many bioportide toxins have a rigid structure with amphiphilic properties important for both cell penetration and bioactivity.

Natural protein sequences are more intrinsically disordered than random sequences.

Most natural protein sequences have resulted from millions or even billions of years of evolution. How they differ from random sequences is not fully understood. Previous computational and experimental studies of random proteins generated from noncoding regions yielded inclusive results due to species-dependent codon biases and GC contents. Here, we approach this problem by investigating 10,000 sequences randomized at the amino acid level. Using well-established predictors for protein intrinsic disorder, we found that natural sequences have more long disordered regions than random sequences, even when random and natural sequences have the same overall composition of amino acid residues. We also showed that random sequences are as structured as natural sequences according to contents and length distributions of predicted secondary structure, although the structures from random sequences may be in a molten globular-like state, according to molecular dynamics simulations. The bias of natural sequences toward more intrinsic disorder suggests that natural sequences are created and evolved to avoid protein aggregation and increase functional diversity.

Effect of structural parameters on the electron capture dissociation and collision-induced dissociation pathways of copper(II)-peptide complexes.

The gas-phase dissociation pathways of proteins/peptides are usually affected by the nature of the charge carrier and the sequence of amino acid residues. The effects of peptide structural parameters, including peptide composition, chain length and amide hydrogen, on the gas-phase dissociation of Cu(II)-model peptide complexes were explored in this study. Polyglycine peptides with flexible frames were used as probes to reduce the complexity of the system and illustrate the mechanism. Results revealed that the types of fragment ions generated in the electron capture dissociation (ECD) of Cu(II)-adducted peptides changed according to the basic amino acid residue composition. Charged or neutral tryptophan side-chain losses were observed in the collision-induced dissociation (CID) of Cu(II)-peptide complexes. Internal electron transfer between tryptophan and metal ion within the complex occurred during the CID reaction, leaving the charge-reduced Cu(+) as a closed d-shell stable electron configuration. The choice of the reaction channel was then determined by the gas-phase basicity of the peptide. Amide hydrogen was critical in the formation of metalated b-/y-ions in the ECD process as determined through mutation of the backbone amide group. Increasing the chain length suppressed the ECD of Cu-metalated peptide species. Our results indicate that the structural parameters of peptides play important roles in the gas-phase dissociation processes of Cu-peptide complexes.

A Novel Cylindrical Representation for Characterizing Intrinsic Properties of Protein Sequences.

The composition and sequence order of amino acid residues are the two most important characteristics to describe a protein sequence. Graphical representations facilitate visualization of biological sequences and produce biologically useful numerical descriptors. In this paper, we propose a novel cylindrical representation by placing the 20 amino acid residue types in a circle and sequence positions along the z axis. This representation allows visualization of the composition and sequence order of amino acids at the same time. Ten numerical descriptors and one weighted numerical descriptor have been developed to quantitatively describe intrinsic properties of protein sequences on the basis of the cylindrical model. Their applications to similarity/dissimilarity analysis of nine ND5 proteins indicated that these numerical descriptors are more effective than several classical numerical matrices. Thus, the cylindrical representation obtained here provides a new useful tool for visualizing and charactering protein sequences. An online server is available at http://biophy.dzu.edu.cn:8080/CNumD/input.jsp .

Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip‐Enhanced Raman Spectroscopy

Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by β-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or α-helical structures, in contrast to the β-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo.

New milk protein-derived peptides with potential antimicrobial activity: an approach based on bioinformatic studies.

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.