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Special Issue "Computational Studies of Biomolecules"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (1 October 2020).

Special Issue Editors

Assoc. Prof. Tatyana Karabencheva-Christova
E-Mail Website
Guest Editor
Department of Chemistry, Michigan Technological University, Houghton, MI 49931, USA
Interests: computational chemical biology; enzyme mechanisms; catalytic activity and inhibition; computer-aided drug design; conformational dynamics of proteins and nucleic acids; biomolecular spectroscopy; bioinorganic enzymology
Special Issues and Collections in MDPI journals
Prof. Dr. Christo Z. Christov
E-Mail Website
Guest Editor

Special Issue Information

Dear Colleagues,

This Special Issue is the continuation of our previous special issue "Computational Studies of Structure-Dynamics-Function Relationships in Biomolecules".

Computational Chemistry Methods are nowadays widely applied for studying biomolecular structure, mechanisms, dynamics, and function. Molecular Dynamic (MD) simulations methods, Quantum Mechanic (QM) methods, Combined Quantum Mechanics/Molecular Mechanics (QM/MM), Molecular Docking and other computational techniques have proven to be very useful for fundamental understanding of structure–function relationships in biomolecules, but also very useful for application in drug design, chemical biology and biotechnology. Importantly, the increased computational power and the development of high-performance computing made further possible the growth in synergistic computational–experimental studies in the most actual areas of biomolecular sciences in timeliness manner.  

The current Special Issue aims to attract high quality contributions of modeling biomolecular structure, dynamics, function and interactions with potential of interpretation of experimental data and application in drug design and protein design.

Topics of interest:

  • Development and validation of new Computational Modeling Methods
  • Computational Studies of proteins structure-function relationships
  • Computational investigations of nucleic acids structure–function relationships
  • Modelling of protein and nucleic acids dynamics
  • Protein Docking
  • Protein-ligand interactions
  • Nucleic acid ligand interactions
  • Protein design
  • Computational enzymology–enzymatic reaction mechanisms
  • Proteins homology modeling

Research Associate Professor Tatyana Karabencheva-Christova
Associate Professor Christo Z. Christov
Guest Editors

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Published Papers (18 papers)

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Article
Alpha-Carbonic Anhydrases from Hydrothermal Vent Sources as Potential Carbon Dioxide Sequestration Agents: In Silico Sequence, Structure and Dynamics Analyses
Int. J. Mol. Sci. 2020, 21(21), 8066; https://doi.org/10.3390/ijms21218066 - 29 Oct 2020
Cited by 1 | Viewed by 793
Abstract
With the increase in CO2 emissions worldwide and its dire effects, there is a need to reduce CO2 concentrations in the atmosphere. Alpha-carbonic anhydrases (α-CAs) have been identified as suitable sequestration agents. This study reports the sequence and structural analysis of [...] Read more.
With the increase in CO2 emissions worldwide and its dire effects, there is a need to reduce CO2 concentrations in the atmosphere. Alpha-carbonic anhydrases (α-CAs) have been identified as suitable sequestration agents. This study reports the sequence and structural analysis of 15 α-CAs from bacteria, originating from hydrothermal vent systems. Structural analysis of the multimers enabled the identification of hotspot and interface residues. Molecular dynamics simulations of the homo-multimers were performed at 300 K, 363 K, 393 K and 423 K to unearth potentially thermostable α-CAs. Average betweenness centrality (BC) calculations confirmed the relevance of some hotspot and interface residues. The key residues responsible for dimer thermostability were identified by comparing fluctuating interfaces with stable ones, and were part of conserved motifs. Crucial long-lived hydrogen bond networks were observed around residues with high BC values. Dynamic cross correlation fortified the relevance of oligomerization of these proteins, thus the importance of simulating them in their multimeric forms. A consensus of the simulation analyses used in this study suggested high thermostability for the α-CA from Nitratiruptor tergarcus. Overall, our novel findings enhance the potential of biotechnology applications through the discovery of alternative thermostable CO2 sequestration agents and their potential protein design. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Molecular Basis for Polyketide Ketoreductase–Substrate Interactions
Int. J. Mol. Sci. 2020, 21(20), 7562; https://doi.org/10.3390/ijms21207562 - 13 Oct 2020
Cited by 1 | Viewed by 603
Abstract
Polyketides are a large class of structurally and functionally diverse natural products with important bioactivities. Many polyketides are synthesized by reducing type II polyketide synthases (PKSs), containing transiently interacting standalone enzymes. During synthesis, ketoreductase (KR) catalyzes regiospecific carbonyl to hydroxyl reduction, determining the [...] Read more.
Polyketides are a large class of structurally and functionally diverse natural products with important bioactivities. Many polyketides are synthesized by reducing type II polyketide synthases (PKSs), containing transiently interacting standalone enzymes. During synthesis, ketoreductase (KR) catalyzes regiospecific carbonyl to hydroxyl reduction, determining the product outcome, yet little is known about what drives specific KR–substrate interactions. In this study, computational approaches were used to explore KR–substrate interactions based on previously solved apo and mimic cocrystal structures. We found five key factors guiding KR–substrate binding. First, two major substrate binding motifs were identified. Second, substrate length is the key determinant of substrate binding position. Third, two key residues in chain length specificity were confirmed. Fourth, phosphorylation of substrates is critical for binding. Finally, packing/hydrophobic effects primarily determine the binding stability. The molecular bases revealed here will help further engineering of type II PKSs and directed biosynthesis of new polyketides. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Mechanisms of Deamidation of Asparagine Residues and Effects of Main-Chain Conformation on Activation Energy
Int. J. Mol. Sci. 2020, 21(19), 7035; https://doi.org/10.3390/ijms21197035 - 24 Sep 2020
Viewed by 875
Abstract
Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and [...] Read more.
Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Understanding the Interaction Modes and Reactivity of Trimedoxime toward MmAChE Inhibited by Nerve Agents: Theoretical and Experimental Aspects
Int. J. Mol. Sci. 2020, 21(18), 6510; https://doi.org/10.3390/ijms21186510 - 05 Sep 2020
Cited by 1 | Viewed by 589
Abstract
Organophosphorus (OP) compounds are used as both chemical weapons and pesticides. However, these agents are very dangerous and toxic to humans, animals, and the environment. Thus, investigations with reactivators have been deeply developed in order to design new antidotes with better efficiency, as [...] Read more.
Organophosphorus (OP) compounds are used as both chemical weapons and pesticides. However, these agents are very dangerous and toxic to humans, animals, and the environment. Thus, investigations with reactivators have been deeply developed in order to design new antidotes with better efficiency, as well as a greater spectrum of action in the acetylcholinesterase (AChE) reactivation process. With that in mind, in this work, we investigated the behavior of trimedoxime toward the Mus musculus acetylcholinesterase (MmAChE) inhibited by a range of nerve agents, such as chemical weapons. From experimental assays, reactivation percentages were obtained for the reactivation of different AChE–OP complexes. On the other hand, theoretical calculations were performed to assess the differences in interaction modes and the reactivity of trimedoxime within the AChE active site. Comparing theoretical and experimental data, it is possible to notice that the oxime, in most cases, showed better reactivation percentages at higher concentrations, with the best result for the reactivation of the AChE–VX adduct. From this work, it was revealed that the mechanistic process contributes most to the oxime efficiency than the interaction in the site. In this way, this study is important to better understand the reactivation process through trimedoxime, contributing to the proposal of novel antidotes. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Plasticity of the 340-Loop in Influenza Neuraminidase Offers New Insight for Antiviral Drug Development
Int. J. Mol. Sci. 2020, 21(16), 5655; https://doi.org/10.3390/ijms21165655 - 06 Aug 2020
Viewed by 671
Abstract
The recently discovered 340-cavity in influenza neuraminidase (NA) N6 and N7 subtypes has introduced new possibilities for rational structure-based drug design. However, the plasticity of the 340-loop (residues 342–347) and the role of the 340-loop in NA activity and substrate binding have not [...] Read more.
The recently discovered 340-cavity in influenza neuraminidase (NA) N6 and N7 subtypes has introduced new possibilities for rational structure-based drug design. However, the plasticity of the 340-loop (residues 342–347) and the role of the 340-loop in NA activity and substrate binding have not been deeply exploited. Here, we investigate the mechanism of 340-cavity formation and demonstrate for the first time that seven of nine NA subtypes are able to adopt an open 340-cavity over 1.8 μs total molecular dynamics simulation time. The finding that the 340-loop plays a role in the sialic acid binding pathway suggests that the 340-cavity can function as a druggable pocket. Comparing the open and closed conformations of the 340-loop, the side chain orientation of residue 344 was found to govern the formation of the 340-cavity. Additionally, the conserved calcium ion was found to substantially influence the stability of the 340-loop. Our study provides dynamical evidence supporting the 340-cavity as a druggable hotspot at the atomic level and offers new structural insight in designing antiviral drugs. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
A Structural Model for Bax∆2-Mediated Activation of Caspase 8-Dependent Apoptosis
Int. J. Mol. Sci. 2020, 21(15), 5476; https://doi.org/10.3390/ijms21155476 - 31 Jul 2020
Viewed by 669
Abstract
Bax∆2 is a pro-apoptotic anti-tumor protein in the Bax family. While most of the Bax family causes cell death by targeting mitochondria, Bax∆2 forms cytosolic aggregates and activates caspase 8-dependent cell death. We previously showed that the Bax∆2 helix α9 is critical for [...] Read more.
Bax∆2 is a pro-apoptotic anti-tumor protein in the Bax family. While most of the Bax family causes cell death by targeting mitochondria, Bax∆2 forms cytosolic aggregates and activates caspase 8-dependent cell death. We previously showed that the Bax∆2 helix α9 is critical for caspase 8 recruitment. However, the interaction between these two proteins at the structural level is unknown. In this in silico study, we performed molecular dynamics (MD) simulations and protein–protein docking on Bax∆2 variants. The results suggest that the Bax∆2 variants have different stable states. Mutating the Baxα mitochondria-targeting signal [L26P/L27P] appears to introduce a kink into helix α1. Protein–protein docking suggests that helices α9 of both wild-type Bax∆2 and Bax∆2 caspase 8 binding-deficient mutant [L164P] can fit in the same caspase 8 binding site, but the mutant is unable to fit as well as wild-type Bax∆2. Together, these data point to a structural basis for explaining Bax∆2 function in caspase 8-dependent cell death. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability
Int. J. Mol. Sci. 2020, 21(14), 4831; https://doi.org/10.3390/ijms21144831 - 08 Jul 2020
Viewed by 762
Abstract
This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the [...] Read more.
This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Activity to Breast Cancer Cell Lines of Different Malignancy and Predicted Interaction with Protein Kinase C Isoforms of Royleanones
Int. J. Mol. Sci. 2020, 21(10), 3671; https://doi.org/10.3390/ijms21103671 - 23 May 2020
Cited by 2 | Viewed by 1529
Abstract
Plants have been used for centuries to treat several illnesses. The Plectranthus genus has a vast variety of species that has allowed the isolation of cytotoxic compounds with notable activities. The abietane diterpenes 6,7-dehydroroyleanone (DeRoy, 1), 7α-acetoxy-6β-hydroxyroyleanone (Roy, 2), and Parvifloron [...] Read more.
Plants have been used for centuries to treat several illnesses. The Plectranthus genus has a vast variety of species that has allowed the isolation of cytotoxic compounds with notable activities. The abietane diterpenes 6,7-dehydroroyleanone (DeRoy, 1), 7α-acetoxy-6β-hydroxyroyleanone (Roy, 2), and Parvifloron D (ParvD, 3) were obtained from Plectranthus spp. and showed promising biological activities, such as cytotoxicity. The inhibitory effects of the different natural abietanes (1-3) were compared in MFC7, SkBr3, and SUM159 cell lines, as well as SUM159 grown in cancer stem cell-inducing conditions. Based on the royleanones’ bioactivity, the derivatives RoyBz (4), RoyBzCl (5), RoyPr2 (6), and DihydroxyRoy (7), previously obtained from 2, were selected for further studies. Protein kinases C (PKCs) are involved in several carcinogenic processes. Thus, PKCs are potential targets for cancer therapy. To date, the portfolio of available PKC modulators remains very limited due to the difficulty of designing isozyme-selective PKC modulators. As such, molecular docking was used to evaluate royleanones 1-6 as predicted isozyme-selective PKC binders. Subtle changes in the binding site of each PKC isoform change the predicted interaction profiles of the ligands. Subtle changes in royleanone substitution patterns, such as a double substitution only with non-substituted phenyls, or hydroxybenzoate at position four that flips the binding mode of ParvD (3), can increase the predicted interactions in certain PKC subtypes. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Discovery of the Novel Inhibitor Against New Delhi Metallo-β-Lactamase Based on Virtual Screening and Molecular Modelling
Int. J. Mol. Sci. 2020, 21(10), 3567; https://doi.org/10.3390/ijms21103567 - 18 May 2020
Cited by 4 | Viewed by 859
Abstract
New Delhi metallo-β-lactamase (NDM-1), one of the metallo-β-lactamases (MBLs), leads to antibiotic resistance in clinical treatments due to the strong ability of hydrolysis to almost all kinds of β-lactam antibiotics. Therefore, there is the urgent need for the research and development of the [...] Read more.
New Delhi metallo-β-lactamase (NDM-1), one of the metallo-β-lactamases (MBLs), leads to antibiotic resistance in clinical treatments due to the strong ability of hydrolysis to almost all kinds of β-lactam antibiotics. Therefore, there is the urgent need for the research and development of the novel drug-resistant inhibitors targeting NDM-1. In this study, ZINC05683641 was screened as potential NDM-1 inhibitor by virtual screening and the inhibitor mechanism of this compound was explored based on molecular dynamics simulation. The nitrocefin assay showed that the IC50 value of ZINC05683641 was 13.59 ± 0.52 μM, indicating that the hydrolytic activity of NDM-1 can be obviously suppressed by ZINC05683641. Further, the binding mode of ZINC05683641 with NDM-1 was obtained by molecular modeling, binding free energy calculation, mutagenesis assays and fluorescence-quenching assays. As results, ILE-35, MET-67, VAL-73, TRP-93, CYS-208, ASN-220 and HIS-250 played the key roles in the binding of NDM-1 with ZINC05683641. Interestingly, these key residues were exactly located in the catalytic activity region of NDM-1, implying that the inhibitor mechanism of ZINC05683641 against NDM-1 was the competitive inhibition. These findings will provide an available approach to research and develop new drug against NDM-1 and treatment for bacterial resistance. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Effects of Single and Double Mutants in Human Glucose-6-Phosphate Dehydrogenase Variants Present in the Mexican Population: Biochemical and Structural Analysis
Int. J. Mol. Sci. 2020, 21(8), 2732; https://doi.org/10.3390/ijms21082732 - 15 Apr 2020
Cited by 2 | Viewed by 1485
Abstract
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to [...] Read more.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to the knowledge of the function and structure of three single natural variants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and a double mutant (G6PD Mount Sinai), each localized in a different region of the three-dimensional (3D) structure. In the functional characterization of the mutants, we observed a decrease in specific activity, protein expression and purification, catalytic efficiency, and substrate affinity in comparison with wild-type (WT) G6PD. Moreover, the analysis of the effect of all mutations on the structural stability showed that its presence increases denaturation and lability with temperature and it is more sensible to trypsin digestion protease and guanidine hydrochloride compared with WT G6PD. This could be explained by accelerated degradation of the variant enzymes due to reduced stability of the protein, as is shown in patients with G6PD deficiency. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Docking and Molecular Dynamics Predictions of Pesticide Binding to the Calyx of Bovine β-Lactoglobulin
Int. J. Mol. Sci. 2020, 21(6), 1988; https://doi.org/10.3390/ijms21061988 - 14 Mar 2020
Viewed by 929
Abstract
Pesticides are used extensively in agriculture, and their residues in food must be monitored to prevent toxicity. The most abundant protein in cow’s milk, β-lactoglobulin (BLG), shows high affinity for diverse hydrophobic ligands in its central binding pocket, called the calyx. Several of [...] Read more.
Pesticides are used extensively in agriculture, and their residues in food must be monitored to prevent toxicity. The most abundant protein in cow’s milk, β-lactoglobulin (BLG), shows high affinity for diverse hydrophobic ligands in its central binding pocket, called the calyx. Several of the most frequently used pesticides are hydrophobic. To predict if BLG may be an unintended carrier for pesticides, we tested its ability to bind 555 pesticides and their isomers, for a total of 889 compounds, in a rigid docking screen. We focused on the analysis of 60 unique molecules belonging to the five pesticide classes defined by the World Health Organization, that docked into BLG’s calyx with ΔGs ranging from −8.2 to −12 kcal mol−1, chosen by statistical criteria. These “potential ligands” were further analyzed using molecular dynamic simulations, and the binding energies were explored with Molecular Mechanics/Generalized Born/Surface Area (MMGBSA). Hydrophobic pyrethroid insecticides, like cypermethrin, were found to bind as deeply and tightly into the calyx as BLG’s natural ligand, palmitate; while polar compounds, like paraquat, were expelled. Our results suggest that BLG could be a carrier for pesticides, in particular for pyrethroid insecticides, allowing for their accumulation in cow’s milk beyond their solubility restrictions. This analysis opens possibilities for pesticide biosensor design based on BLG. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Ligand Binding Mechanism and Its Relationship with Conformational Changes in Adenine Riboswitch
Int. J. Mol. Sci. 2020, 21(6), 1926; https://doi.org/10.3390/ijms21061926 - 11 Mar 2020
Cited by 3 | Viewed by 919
Abstract
Riboswitches are naturally occurring RNA aptamers that control the expression of essential bacterial genes by binding to specific small molecules. The binding with both high affinity and specificity induces conformational changes. Thus, riboswitches were proposed as a possible molecular target for developing antibiotics [...] Read more.
Riboswitches are naturally occurring RNA aptamers that control the expression of essential bacterial genes by binding to specific small molecules. The binding with both high affinity and specificity induces conformational changes. Thus, riboswitches were proposed as a possible molecular target for developing antibiotics and chemical tools. The adenine riboswitch can bind not only to purine analogues but also to pyrimidine analogues. Here, long molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) computational methodologies were carried out to show the differences in the binding model and the conformational changes upon five ligands binding. The binding free energies of the guanine riboswitch aptamer with C74U mutation complexes were compared to the binding free energies of the adenine riboswitch (AR) aptamer complexes. The calculated results are in agreement with the experimental data. The differences for the same ligand binding to two different aptamers are related to the electrostatic contribution. Binding dynamical analysis suggests a flexible binding pocket for the pyrimidine ligand in comparison with the purine ligand. The 18 μs of MD simulations in total indicate that both ligand-unbound and ligand-bound aptamers transfer their conformation between open and closed states. The ligand binding obviously affects the conformational change. The conformational states of the aptamer are associated with the distance between the mass center of two key nucleotides (U51 and A52) and the mass center of the other two key nucleotides (C74 and C75). The results suggest that the dynamical character of the binding pocket would affect its biofunction. To design new ligands of the adenine riboswitch, it is recommended to consider the binding affinities of the ligand and the conformational change of the ligand binding pocket. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Elucidating the Structural Basis of the Intracellular pH Sensing Mechanism of TASK-2 K2P Channels
Int. J. Mol. Sci. 2020, 21(2), 532; https://doi.org/10.3390/ijms21020532 - 14 Jan 2020
Cited by 2 | Viewed by 1359
Abstract
Two-pore domain potassium (K2P) channels maintain the cell’s background conductance by stabilizing the resting membrane potential. They assemble as dimers possessing four transmembrane helices in each subunit. K2P channels were crystallized in “up” and “down” states. The movements of [...] Read more.
Two-pore domain potassium (K2P) channels maintain the cell’s background conductance by stabilizing the resting membrane potential. They assemble as dimers possessing four transmembrane helices in each subunit. K2P channels were crystallized in “up” and “down” states. The movements of the pore-lining transmembrane TM4 helix produce the aperture or closure of side fenestrations that connect the lipid membrane with the central cavity. When the TM4 helix is in the up-state, the fenestrations are closed, while they are open in the down-state. It is thought that the fenestration states are related to the activity of K2P channels and the opening of the channels preferentially occurs from the up-state. TASK-2, a member of the TALK subfamily of K2P channels, is opened by intracellular alkalization leading the deprotonation of the K245 residue at the end of the TM4 helix. This charge neutralization of K245 could be sensitive or coupled to the fenestration state. Here, we describe the relationship between the states of the intramembrane fenestrations and K245 residue in TASK-2 channel. By using molecular modeling and simulations, we show that the protonated state of K245 (K245+) favors the open fenestration state and, symmetrically, that the open fenestration state favors the protonated state of the lysine residue. We show that the channel can be completely blocked by Prozac, which is known to induce fenestration opening in TREK-2. K245 protonation and fenestration aperture have an additive effect on the conductance of the channel. The opening of the fenestrations with K245+ increases the entrance of lipids into the selectivity filter, blocking the channel. At the same time, the protonation of K245 introduces electrostatic potential energy barriers to ion entrance. We computed the free energy profiles of ion penetration into the channel in different fenestration and K245 protonation states, to show that the effects of the two transformations are summed up, leading to maximum channel blocking. Estimated rates of ion transport are in qualitative agreement with experimental results and support the hypothesis that the most important barrier for ion transport under K245+ and open fenestration conditions is the entrance of the ions into the channel. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Structural Characterization of the CD44 Stem Region for Standard and Cancer-Associated Isoforms
Int. J. Mol. Sci. 2020, 21(1), 336; https://doi.org/10.3390/ijms21010336 - 03 Jan 2020
Cited by 1 | Viewed by 1556
Abstract
CD44 is widely expressed in most vertebrate cells, whereas the expression of CD44v6 is restricted to only a few tissues and has been considered to be associated with tumor progression and metastasis. Thus, CD44v6 has been recognized as a promising prognostic biomarker and [...] Read more.
CD44 is widely expressed in most vertebrate cells, whereas the expression of CD44v6 is restricted to only a few tissues and has been considered to be associated with tumor progression and metastasis. Thus, CD44v6 has been recognized as a promising prognostic biomarker and therapeutic target for various cancers for more than a decade. However, despite many experimental studies, the structural dynamics and differences between CD44s and CD44v6, particularly in their stem region, still remain elusive. Here, a computational study was conducted to address these problems. We found that the stem of CD44s adopted predominantly two conformations, one featuring antiparallel β-sheets and the other featuring parallel β-sheets, whereas the stem of CD44v6 adopted mainly one conformation with relatively highly suppressed β-sheet contents. Moreover, Phe215 was found to be essential in the β-sheets of both CD44s and CD44v6. We finally found intramolecular Phe215–Trp224 hydrogen-bonding interactions and hydrophobic interactions with Phe215 that cooperatively drove conformational differences upon the addition of the v6 region to CD44. Our study elucidated the structural differences between the stem regions of CD44s and CD44v6 and thus can offer useful structural information for drug design to specifically target CD44v6 in promising clinical applications. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Novel Descriptors and Digital Signal Processing- Based Method for Protein Sequence Activity Relationship Study
Int. J. Mol. Sci. 2019, 20(22), 5640; https://doi.org/10.3390/ijms20225640 - 11 Nov 2019
Cited by 1 | Viewed by 1448
Abstract
The work aiming to unravel the correlation between protein sequence and function in the absence of structural information can be highly rewarding. We present a new way of considering descriptors from the amino acids index database for modeling and predicting the fitness value [...] Read more.
The work aiming to unravel the correlation between protein sequence and function in the absence of structural information can be highly rewarding. We present a new way of considering descriptors from the amino acids index database for modeling and predicting the fitness value of a polypeptide chain. This approach includes the following steps: (i) Calculating Q elementary numerical sequences (Ele_SEQ) depending on the encoding of the amino acid residues, (ii) determining an extended numerical sequence (Ext_SEQ) by concatenating the Q elementary numerical sequences, wherein at least one elementary numerical sequence is a protein spectrum obtained by applying fast Fourier transformation (FFT), and (iii) predicting a value of fitness for polypeptide variants (train and/or validation set). These new descriptors were tested on four sets of proteins of different lengths (GLP-2, TNF alpha, cytochrome P450, and epoxide hydrolase) and activities (cAMP activation, binding affinity, thermostability and enantioselectivity). We show that the use of multiple physicochemical descriptors coupled with the implementation of the FFT, taking into account the interactions between residues of amino acids within the protein sequence, could lead to very significant improvement in the quality of models and predictions. The choice of the descriptor or of the combination of descriptors and/or FFT is dependent on the couple protein/fitness. This approach can provide potential users with value added to existing mutant libraries where screening efforts have so far been unsuccessful in finding improved polypeptide mutants for useful applications. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Molecular Cloning and Exploration of the Biochemical and Functional Analysis of Recombinant Glucose-6-Phosphate Dehydrogenase from Gluconoacetobacter diazotrophicus PAL5
Int. J. Mol. Sci. 2019, 20(21), 5279; https://doi.org/10.3390/ijms20215279 - 24 Oct 2019
Cited by 2 | Viewed by 1206
Abstract
Gluconacetobacter diazotrophicus PAL5 (GDI) is an endophytic bacterium with potential biotechnological applications in industry and agronomy. The recent description of its complete genome and its principal metabolic enzymes suggests that glucose metabolism is accomplished through the pentose phosphate pathway (PPP); however, the enzymes [...] Read more.
Gluconacetobacter diazotrophicus PAL5 (GDI) is an endophytic bacterium with potential biotechnological applications in industry and agronomy. The recent description of its complete genome and its principal metabolic enzymes suggests that glucose metabolism is accomplished through the pentose phosphate pathway (PPP); however, the enzymes participating in this pathway have not yet been characterized in detail. The objective of the present work was to clone, purify, and biochemically and physicochemically characterize glucose-6-phosphate dehydrogenase (G6PD) from GDI. The gene was cloned and expressed as a tagged protein in E. coli to be purified by affinity chromatography. The native state of the G6PD protein in the solution was found to be a tetramer with optimal activity at pH 8.8 and a temperature between 37 and 50 °C. The apparent Km values for G6P and nicotinamide adenine dinucleotide phosphate (NADP+) were 63 and 7.2 μM, respectively. Finally, from the amino acid sequence a three-dimensional (3D) model was obtained, which allowed the arrangement of the amino acids involved in the catalytic activity, which are conserved (RIDHYLGKE, GxGGDLT, and EKPxG) with those of other species, to be identified. This characterization of the enzyme could help to identify new environmental conditions for the knowledge of the plant–microorganism interactions and a better use of GDI in new technological applications. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Article
Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP
Int. J. Mol. Sci. 2019, 20(20), 5229; https://doi.org/10.3390/ijms20205229 - 22 Oct 2019
Cited by 7 | Viewed by 1118
Abstract
Enhanced green fluorescent protein (EGFP)—one of the most widely applied genetically encoded fluorescent probes—carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion (“redding”) with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a [...] Read more.
Enhanced green fluorescent protein (EGFP)—one of the most widely applied genetically encoded fluorescent probes—carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion (“redding”) with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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Review

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Review
Dynamics, a Powerful Component of Current and Future in Silico Approaches for Protein Design and Engineering
Int. J. Mol. Sci. 2020, 21(8), 2713; https://doi.org/10.3390/ijms21082713 - 14 Apr 2020
Cited by 4 | Viewed by 1799
Abstract
Computational prediction has become an indispensable aid in the processes of engineering and designing proteins for various biotechnological applications. With the tremendous progress in more powerful computer hardware and more efficient algorithms, some of in silico tools and methods have started to apply [...] Read more.
Computational prediction has become an indispensable aid in the processes of engineering and designing proteins for various biotechnological applications. With the tremendous progress in more powerful computer hardware and more efficient algorithms, some of in silico tools and methods have started to apply the more realistic description of proteins as their conformational ensembles, making protein dynamics an integral part of their prediction workflows. To help protein engineers to harness benefits of considering dynamics in their designs, we surveyed new tools developed for analyses of conformational ensembles in order to select engineering hotspots and design mutations. Next, we discussed the collective evolution towards more flexible protein design methods, including ensemble-based approaches, knowledge-assisted methods, and provable algorithms. Finally, we highlighted apparent challenges that current approaches are facing and provided our perspectives on their further development. Full article
(This article belongs to the Special Issue Computational Studies of Biomolecules)
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