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Topical Collection "Feature Papers in Molecular Biophysics"

Editors

Prof. Dr. Ian A. Nicholls
E-Mail Website
Collection Editor
Department of Chemistry and Biomedical Sciences, Linnaeus University, Kalmar, Sweden
Interests: biomimetic systems; complex mixture modelling and spectroscopic studies of biological; synthetic and hybrid systems
Special Issues and Collections in MDPI journals
Dr. Vladimir N. Uversky
grade E-Mail Website
Collection Editor
Department of Molecular Medicine, USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., MDC07, Tampa, FL 33612, USA
Interests: intrinsically disordered proteins; protein folding; protein misfolding; partially folded proteins; protein aggregation; protein structure; protein function; protein stability; protein biophysics; protein bioinformatics; conformational diseases; protein–ligand interactions; protein–protein interactions; liquid-liquid phase transitions
Special Issues and Collections in MDPI journals

Topical Collection Information

Dear Colleagues,

As follows from the title, this Topical Collection “Feature Papers in Molecular Biophysics” aims to collect high quality research articles, short communications, and review articles in all the fields of molecular biophysics. Since the aim of this Topical Collection is to illustrate, through selected works, frontier research in molecular biophysics, we encourage Editorial Board Members of the Molecular Biophysics Section of the International Journal of Molecular Sciences to contribute papers reflecting the latest progress in their research field, or to invite relevant experts and colleagues to do so.

Topics include, but are not limited to:

  • molecular structure and dynamics
  • nucleic acid structure and dynamics
  • protein structure and dynamics
  • membrane structure and dynamics
  • biomimetic material structure and dynamics
  • molecular simulations
  • molecular modeling
  • single molecule biophysics
  • biophysical techniques in the study of biomacromolecular and biomimetic systems
  • biomolecular interactions
  • biomimetic material interactions
  • macromolecular structure determination or prediction
  • characterization of disordered proteins and their interactions
  • computational biophysics
  • bioinformatics
  • biophysical and computational approaches to drug design and development
  • advances in molecular biophysical methodologies as well as imaging techniques and data analysis
  • application of biophysical methods

Prof. Dr. Ian A. Nicholls
Dr. Vladimir N. Uversky
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

  • biophysics
  • molecular imprinting
  • molecular simulations
  • molecular structure
  • molecular dynamics
  • molecular mechanics
  • thermodynamics
  • biomolecular interactions
  • protein structure and folding
  • intrinsically disordered proteins
  • structure prediction
  • nucleic acid–protein interactions
  • protein–membrane interactions
  • protein–DNA interactions
  • posttranslational modifications
  • drug–receptor interactions
  • protein design
  • protein engineering
  • protein–ligand binding
  • transmembrane proteins
  • chaperones
  • enzymology
  • molecular recognition
  • molecular modeling
  • membrane dynamics
  • macromolecular structure and dynamics
  • DNA structure and dynamics
  • RNA structure
  • genome structure
  • structure–function relationships
  • ion channels
  • spectroscopic techniques
  • biomolecular NMR
  • inter-molecular interactions
  • X-ray crystallography
  • macromolecular crystallography
  • crystal thermodynamics
  • microcalorimetry
  • transient kinetic techniques
  • fluorescence imaging
  • single-molecule microscopy
  • statistical mechanics
  • computer simulations
  • computational modeling
  • molecular modeling

Published Papers (59 papers)

2021

Jump to: 2020, 2019

Review
Lipid Self-Assemblies under the Atomic Force Microscope
Int. J. Mol. Sci. 2021, 22(18), 10085; https://doi.org/10.3390/ijms221810085 - 18 Sep 2021
Viewed by 194
Abstract
Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular [...] Read more.
Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols…), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling. Full article
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Article
Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure
Int. J. Mol. Sci. 2021, 22(18), 10012; https://doi.org/10.3390/ijms221810012 - 16 Sep 2021
Viewed by 305
Abstract
Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was [...] Read more.
Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment. Full article
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Review
An Overview of Near Infrared Spectroscopy and Its Applications in the Detection of Genetically Modified Organisms
Int. J. Mol. Sci. 2021, 22(18), 9940; https://doi.org/10.3390/ijms22189940 - 14 Sep 2021
Viewed by 290
Abstract
Near-infrared spectroscopy (NIRS) has become a more popular approach for quantitative and qualitative analysis of feeds, foods and medicine in conjunction with an arsenal of chemometric tools. This was the foundation for the increased importance of NIRS in other fields, like genetics and [...] Read more.
Near-infrared spectroscopy (NIRS) has become a more popular approach for quantitative and qualitative analysis of feeds, foods and medicine in conjunction with an arsenal of chemometric tools. This was the foundation for the increased importance of NIRS in other fields, like genetics and transgenic monitoring. A considerable number of studies have utilized NIRS for the effective identification and discrimination of plants and foods, especially for the identification of genetically modified crops. Few previous reviews have elaborated on the applications of NIRS in agriculture and food, but there is no comprehensive review that compares the use of NIRS in the detection of genetically modified organisms (GMOs). This is particularly important because, in comparison to previous technologies such as PCR and ELISA, NIRS offers several advantages, such as speed (eliminating time-consuming procedures), non-destructive/non-invasive analysis, and is inexpensive in terms of cost and maintenance. More importantly, this technique has the potential to measure multiple quality components in GMOs with reliable accuracy. In this review, we brief about the fundamentals and versatile applications of NIRS for the effective identification of GMOs in the agricultural and food systems. Full article
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Article
Muscimol Directly Activates the TREK-2 Channel Expressed in GABAergic Neurons through Its N-Terminus
Int. J. Mol. Sci. 2021, 22(17), 9320; https://doi.org/10.3390/ijms22179320 - 27 Aug 2021
Viewed by 383
Abstract
The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular [...] Read more.
The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons. Full article
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Article
Decreased Interactions between Calmodulin and a Mutant Huntingtin Model Might Reduce the Cytotoxic Level of Intracellular Ca2+: A Molecular Dynamics Study
Int. J. Mol. Sci. 2021, 22(16), 9025; https://doi.org/10.3390/ijms22169025 - 21 Aug 2021
Viewed by 457
Abstract
Mutant huntingtin (m-HTT) proteins and calmodulin (CaM) co-localize in the cerebral cortex with significant effects on the intracellular calcium levels by altering the specific calcium-mediated signals. Furthermore, the mutant huntingtin proteins show great affinity for CaM that can lead to a further stabilization [...] Read more.
Mutant huntingtin (m-HTT) proteins and calmodulin (CaM) co-localize in the cerebral cortex with significant effects on the intracellular calcium levels by altering the specific calcium-mediated signals. Furthermore, the mutant huntingtin proteins show great affinity for CaM that can lead to a further stabilization of the mutant huntingtin aggregates. In this context, the present study focuses on describing the interactions between CaM and two huntingtin mutants from a biophysical point of view, by using classical Molecular Dynamics techniques. The huntingtin models consist of a wild-type structure, one mutant with 45 glutamine residues and the second mutant with nine additional key-point mutations from glutamine residues into proline residues (9P(EM) model). Our docking scores and binding free energy calculations show higher binding affinities of all HTT models for the C-lobe end of the CaM protein. In terms of dynamic evolution, the 9P(EM) model triggered great structural changes into the CaM protein’s structure and shows the highest fluctuation rates due to its structural transitions at the helical level from α-helices to turns and random coils. Moreover, our proposed 9P(EM) model suggests much lower interaction energies when compared to the 45Qs-HTT mutant model, this finding being in good agreement with the 9P(EM)’s antagonistic effect hypothesis on highly toxic protein–protein interactions. Full article
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Article
BRAF Modulates Stretch-Induced Intercellular Gap Formation through Localized Actin Reorganization
Int. J. Mol. Sci. 2021, 22(16), 8989; https://doi.org/10.3390/ijms22168989 - 20 Aug 2021
Viewed by 306
Abstract
Mechanical forces acting on cell–cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell–cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the [...] Read more.
Mechanical forces acting on cell–cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell–cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the stress-induced response. Control cells responded to external forces by organizing and stabilizing actin cables in the stretched cell junctions. This was accompanied by an increase in intercellular gap formation, which was prevented in BRAF knockdown monolayers. In the absence of BRAF, there was excess stress fiber formation due to the enhanced reorganization of actin fibers. Our findings suggest that stretch-induced intercellular gap formation, leading to a decrease in barrier function of blood vessels, can be reverted by BRAF RNAi. This is important when the endothelium experiences changes in external stresses caused by high blood pressure, leading to edema, or by immune or cancer cells in inflammation or metastasis. Full article
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Review
Application of Monolayer Graphene and Its Derivative in Cryo-EM Sample Preparation
by , , and
Int. J. Mol. Sci. 2021, 22(16), 8940; https://doi.org/10.3390/ijms22168940 - 19 Aug 2021
Viewed by 368
Abstract
Cryo-electron microscopy (Cryo-EM) has become a routine technology for resolving the structure of biological macromolecules due to the resolution revolution in recent years. The specimens are typically prepared in a very thin layer of vitrified ice suspending in the holes of the perforated [...] Read more.
Cryo-electron microscopy (Cryo-EM) has become a routine technology for resolving the structure of biological macromolecules due to the resolution revolution in recent years. The specimens are typically prepared in a very thin layer of vitrified ice suspending in the holes of the perforated amorphous carbon film. However, the samples prepared by directly applying to the conventional support membranes may suffer from partial or complete denaturation caused by sticking to the air–water interface (AWI). With the application in materials, graphene has also been used recently to improve frozen sample preparation instead of a suspended conventional amorphous thin carbon. It has been proven that graphene or graphene oxide and various chemical modifications on its surface can effectively prevent particles from adsorbing to the AWI, which improves the dispersion, adsorbed number, and orientation preference of frozen particles in the ice layer. Their excellent properties and thinner thickness can significantly reduce the background noise, allowing high-resolution three-dimensional reconstructions using a minimum data set. Full article
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Article
High Power Electromagnetic Waves Exposure of Healthy and Tumor Bearing Mice: Assessment of Effects on Mice Growth, Behavior, Tumor Growth, and Vessel Permeabilization
Int. J. Mol. Sci. 2021, 22(16), 8516; https://doi.org/10.3390/ijms22168516 - 07 Aug 2021
Viewed by 385
Abstract
High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects [...] Read more.
High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects upon exposure of healthy and tumor-bearing mice. In field experiments, the exposure to 1.5 GHz narrow-band electromagnetic fields with the incident amplitude peak value level in the range of 40 kV/m and 150 MHz wide-band electric fields with the amplitude peak value in the range of 200 kV/m, did not alter healthy and tumor-bearing animals’ growth, nor it had any impact on cutaneous murine tumors’ growth. While we did not observe any noticeable behavioral changes in mice during the exposure to narrow-band signals when wide-band HPEM signals were applied, mice could behave in a similar way as they respond to loud noise signals: namely, if a mouse was exploring the cage prior to signal application, it returned to companion mates when wide-band HPEM signals were applied. Moreover, the effect of wide-band signals was assessed on normal blood vessels permeability in real-time in dorsal-chamber-bearing mice exposed in a pilot study using wide-band signal applicators. Our pilot study conducted within the applicator and performed at the laboratory scale suggests that the exposure to wide-band signals with the amplitude of 47.5 kV/m does not result in increased vessel permeability. Full article
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Article
Effects of Visfatin on Intracellular Mechanics and Catabolism in Human Primary Chondrocytes through Glycogen Synthase Kinase 3β Inactivation
Int. J. Mol. Sci. 2021, 22(15), 8107; https://doi.org/10.3390/ijms22158107 - 28 Jul 2021
Viewed by 484
Abstract
Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant [...] Read more.
Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant attention because of the clinical finding of the positive correlation between its serum/synovial level and OA progression. However, the precise mechanism involved is still unclear. This study determined the effect of visfatin on intracellular mechanics and catabolism in human primary chondrocytes isolated from patients. The intracellular stiffness of chondrocytes was analyzed by the particle-tracking microrheology method. It was shown that visfatin damages the microtubule and microfilament networks to influence intracellular mechanics to decrease the intracellular elasticity and viscosity via glycogen synthase kinase 3β (GSK3β) inactivation induced by p38 signaling. Further, microtubule network destruction in human primary chondrocytes is predominantly responsible for the catabolic effect of visfatin on the cyclooxygenase 2 upregulation. The present study shows a more comprehensive interpretation of OA development induced by visfatin through biochemical and biophysical perspectives. Finally, the role of GSK3β inactivation, and subsequent regulation of intracellular mechanics, might be considered as theranostic targets for future drug development for OA. Full article
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Article
Effect of Dimer Structure and Inhomogeneous Broadening of Energy Levels on the Action of Flavomononucleotide in Rigid Polyvinyl Alcohol Films
Int. J. Mol. Sci. 2021, 22(14), 7759; https://doi.org/10.3390/ijms22147759 - 20 Jul 2021
Viewed by 507
Abstract
The results of time-resolved fluorescence measurements of flavin mononucleotide (FMN) in rigid polyvinyl alcohol films (PVA) demonstrate that fluorescence intensity decays are strongly accelerated in the presence of fluorescent dimers and nonradiative energy transfer processes. The fluorescence decay originating both from H and [...] Read more.
The results of time-resolved fluorescence measurements of flavin mononucleotide (FMN) in rigid polyvinyl alcohol films (PVA) demonstrate that fluorescence intensity decays are strongly accelerated in the presence of fluorescent dimers and nonradiative energy transfer processes. The fluorescence decay originating both from H and J dimer states of FMN was experimentally observed for the first time. The mean fluorescence lifetimes for FMN dimers were obtained: τfl = 2.66 ns (at λexc = 445 nm) and τfl = 2.02 (at λexc = 487 nm) at λobs = 600 nm and T = 253 K from H and J state of dimers, respectively. We show that inhomogeneous orientational broadening of energy levels (IOBEL) affects the shape of the fluorescence decay and leads to the dependence of the average monomer fluorescence lifetime on excitation wavelength. IOBEL affected the nonradiative energy transfer and indicated that different flavin positioning in the protein pocket could (1) change the spectroscopic properties of flavins due to the existence of “blue” and “red” fluorescence centers, and (2) diminish the effectiveness of energy transfer between FMN molecules. Full article
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Article
The Mode of SN38 Derivatives Interacting with Nicked DNA Mimics Biological Targeting of Topo I Poisons
Int. J. Mol. Sci. 2021, 22(14), 7471; https://doi.org/10.3390/ijms22147471 - 12 Jul 2021
Viewed by 580
Abstract
The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered [...] Read more.
The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI–MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the 1H/13C HSQC spectrum of 13C-enriched compound 3. In this work, we showed that the studied compounds—parent compounds 2 and 3, and their potential metabolite 4 and SN38—interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity. Full article
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Review
Human Radiosensitivity and Radiosusceptibility: What Are the Differences?
Int. J. Mol. Sci. 2021, 22(13), 7158; https://doi.org/10.3390/ijms22137158 - 02 Jul 2021
Viewed by 738
Abstract
The individual response to ionizing radiation (IR) raises a number of medical, scientific, and societal issues. While the term “radiosensitivity” was used by the pioneers at the beginning of the 20st century to describe only the radiation-induced adverse tissue reactions related to cell [...] Read more.
The individual response to ionizing radiation (IR) raises a number of medical, scientific, and societal issues. While the term “radiosensitivity” was used by the pioneers at the beginning of the 20st century to describe only the radiation-induced adverse tissue reactions related to cell death, a confusion emerged in the literature from the 1930s, as “radiosensitivity” was indifferently used to describe the toxic, cancerous, or aging effect of IR. In parallel, the predisposition to radiation-induced adverse tissue reactions (radiosensitivity), notably observed after radiotherapy appears to be caused by different mechanisms than those linked to predisposition to radiation-induced cancer (radiosusceptibility). This review aims to document these differences in order to better estimate the different radiation-induced risks. It reveals that there are very few syndromes associated with the loss of biological functions involved directly in DNA damage recognition and repair as their role is absolutely necessary for cell viability. By contrast, some cytoplasmic proteins whose functions are independent of genome surveillance may also act as phosphorylation substrates of the ATM protein to regulate the molecular response to IR. The role of the ATM protein may help classify the genetic syndromes associated with radiosensitivity and/or radiosusceptibility. Full article
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Article
Engineering of Janus-Like Dendrimers with Peptides Derived from Glycoproteins of Herpes Simplex Virus Type 1: Toward a Versatile and Novel Antiviral Platform
Int. J. Mol. Sci. 2021, 22(12), 6488; https://doi.org/10.3390/ijms22126488 - 17 Jun 2021
Cited by 2 | Viewed by 557
Abstract
Novel antiviral nanotherapeutics, which may inactivate the virus and block it from entering host cells, represent an important challenge to face viral global health emergencies around the world. Using a combination of bioorthogonal copper-catalyzed 1,3-dipolar alkyne/azide cycloaddition (CuAAC) and photoinitiated thiol–ene coupling, monofunctional [...] Read more.
Novel antiviral nanotherapeutics, which may inactivate the virus and block it from entering host cells, represent an important challenge to face viral global health emergencies around the world. Using a combination of bioorthogonal copper-catalyzed 1,3-dipolar alkyne/azide cycloaddition (CuAAC) and photoinitiated thiol–ene coupling, monofunctional and bifunctional peptidodendrimer conjugates were obtained. The conjugates are biocompatible and demonstrate no toxicity to cells at biologically relevant concentrations. Furthermore, the orthogonal addition of multiple copies of two different antiviral peptides on the surface of a single dendrimer allowed the resulting bioconjugates to inhibit Herpes simplex virus type 1 at both the early and the late stages of the infection process. The presented work builds on further improving this attractive design to obtain a new class of therapeutics. Full article
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Article
A Glimpse into the Structural Properties of the Intermediate and Transition State in the Folding of Bromodomain 2 Domain 2 by Φ Value Analysis
Int. J. Mol. Sci. 2021, 22(11), 5953; https://doi.org/10.3390/ijms22115953 - 31 May 2021
Viewed by 649
Abstract
Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. [...] Read more.
Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. BRD inhibition is considered an attractive therapeutic approach in epigenetic disorders, particularly in oncology. Here, we present a Φ value analysis to investigate the folding pathway of the second domain of BRD2 (BRD2(2)). Using an extensive mutational analysis based on 25 site-directed mutants, we provide structural information on both the intermediate and late transition state of BRD2(2). The data reveal that the C-terminal region represents part of the initial folding nucleus, while the N-terminal region of the domain consolidates its structure only later in the folding process. Furthermore, only a small number of native-like interactions have been identified, suggesting the presence of a non-compact, partially folded state with scarce native-like characteristics. Taken together, these results indicate that, in BRD2(2), a hierarchical mechanism of protein folding can be described with non-native interactions that play a significant role in folding. Full article
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Article
Serotonin Promotes Serum Albumin Interaction with the Monomeric Amyloid β Peptide
Int. J. Mol. Sci. 2021, 22(11), 5896; https://doi.org/10.3390/ijms22115896 - 31 May 2021
Viewed by 760
Abstract
Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, [...] Read more.
Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aβ by a factor of 3 (BBRC, 510(2), 248–253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aβ monomer to HSA by a factor of 7/17 for Aβ40/Aβ42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA’s affinity to monomeric Aβ, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aβ release from HSA in the central nervous system due to impairment of the SRO-mediated Aβ trapping by HSA. Full article
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Article
KEAP1 Cancer Mutants: A Large-Scale Molecular Dynamics Study of Protein Stability
Int. J. Mol. Sci. 2021, 22(10), 5408; https://doi.org/10.3390/ijms22105408 - 20 May 2021
Viewed by 994
Abstract
We have performed 280 μs of unbiased molecular dynamics (MD) simulations to investigate the effects of 12 different cancer mutations on Kelch-like ECH-associated protein 1 (KEAP1) (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C, R470C, R470H, R470S and G476R), one of the [...] Read more.
We have performed 280 μs of unbiased molecular dynamics (MD) simulations to investigate the effects of 12 different cancer mutations on Kelch-like ECH-associated protein 1 (KEAP1) (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C, R470C, R470H, R470S and G476R), one of the frequently mutated proteins in lung cancer. The aim was to provide structural insight into the effects of these mutants, including a new class of ANCHOR (additionally NRF2-complexed hypomorph) mutant variants. Our work provides additional insight into the structural dynamics of mutants that could not be analyzed experimentally, painting a more complete picture of their mutagenic effects. Notably, blade-wise analysis of the Kelch domain points to stability as a possible target of cancer in KEAP1. Interestingly, structural analysis of the R470C ANCHOR mutant, the most prevalent missense mutation in KEAP1, revealed no significant change in structural stability or NRF2 binding site dynamics, possibly indicating an covalent modification as this mutant’s mode of action. Full article
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Article
The Study on Molecular Profile Changes of Pathogens via Zinc Nanocomposites Immobilization Approach
Int. J. Mol. Sci. 2021, 22(10), 5395; https://doi.org/10.3390/ijms22105395 - 20 May 2021
Viewed by 558
Abstract
The most critical group of all includes multidrug resistant bacteria that pose a particular threat in hospitals, as they can cause severe and often deadly infections. Modern medicine still faces the difficult task of developing new agents for the effective control of bacterial-based [...] Read more.
The most critical group of all includes multidrug resistant bacteria that pose a particular threat in hospitals, as they can cause severe and often deadly infections. Modern medicine still faces the difficult task of developing new agents for the effective control of bacterial-based diseases. The targeted administration of nanoparticles can enhance the efficiency of conventional pharmaceutical agents. However, the interpretation of interfaces’ interactions between nanoparticles and biological systems still remains a challenge for researchers. In fact, the current research presents a strategy for using ZnO NPs immobilization with ampicillin and tetracycline. Firstly, the study provides the mechanism of the ampicillin and tetracycline binding on the surface of ZnO NPs. Secondly, it examines the effect of non-immobilized ZnO NPs, immobilized with ampicillin (ZnONPs/AMP) and tetracycline (ZnONPs/TET), on the cells’ metabolism and morphology, based on the protein and lipid profiles. A sorption kinetics study showed that the antibiotics binding on the surface of ZnONPs depend on their structure. The efficiency of the process was definitely higher in the case of ampicillin. In addition, flow cytometry results showed that immobilized nanoparticles present a different mechanism of action. Moreover, according to the MALDI approach, the antibacterial activity mechanism of the investigated ZnO complexes is mainly based on the destruction of cell membrane integrity by lipids and proteins, which is necessary for proper cell function. Additionally, it was noticed that some of the identified changes indicate the activation of defense mechanisms by cells, leading to a decrease in the permeability of a cell’s external barriers or the synthesis of repair proteins. Full article
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Article
Some Dietary Phenolic Compounds Can Activate Thyroid Peroxidase and Inhibit Lipoxygenase-Preliminary Study in the Model Systems
Int. J. Mol. Sci. 2021, 22(10), 5108; https://doi.org/10.3390/ijms22105108 - 12 May 2021
Viewed by 457
Abstract
The presented research concerns the triple activity of trans-cinnamic (tCA), ferulic (FA) and syringic acids (SA). They act as thyroid peroxidase (TPO) activators, lipoxygenase (LOX) inhibitors and show antiradical activity. All compounds showed a dose-dependent TPO activatory effect, thus the AC50 [...] Read more.
The presented research concerns the triple activity of trans-cinnamic (tCA), ferulic (FA) and syringic acids (SA). They act as thyroid peroxidase (TPO) activators, lipoxygenase (LOX) inhibitors and show antiradical activity. All compounds showed a dose-dependent TPO activatory effect, thus the AC50 value (the concentration resulting in 50% activation) was determined. The tested compounds can be ranked as follows: tCA > FA > SA with AC50 = 0.10, 0.39, 0.69 mM, respectively. Strong synergism was found between FA and SA. The activatory effects of all tested compounds may result from interaction with the TPO allosteric site. It was proposed that conformational change resulting from activator binding to TPO allosteric pocket results from the flexibility of a nearby loop formed by residues Val352-Tyr363. All compounds act as uncompetitive LOX inhibitors. The most effective were tCA and SA, whereas the weakest was FA (IC50 = 0.009 mM and IC50 0.027 mM, respectively). In all cases, an interaction between the inhibitors carboxylic groups and side-chain atoms of Arg102 and Arg139 in an allosteric pocket of LOX was suggested. FA/tCA and FA/SA acted synergistically, whereas tCA/SA demonstrated antagonism. The highest antiradical activity was found in the case of SA (IC50 = 0.22 mM). FA/tCA and tCA/SA acted synergistically, whereas antagonism was found for the SA/FA mixture. Full article
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Article
Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers
Int. J. Mol. Sci. 2021, 22(9), 4682; https://doi.org/10.3390/ijms22094682 - 28 Apr 2021
Viewed by 628
Abstract
Background: Cancer stem cells’ (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs’ subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. [...] Read more.
Background: Cancer stem cells’ (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs’ subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. Methods: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). Results: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. Conclusion: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment. Full article
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Article
Red-Edge Excitation Shift Spectroscopy (REES): Application to Hidden Bound States of Ligands in Protein–Ligand Complexes
Int. J. Mol. Sci. 2021, 22(5), 2582; https://doi.org/10.3390/ijms22052582 - 04 Mar 2021
Viewed by 597
Abstract
Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can [...] Read more.
Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein–ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein–ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor–kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor–kinase interactions. Full article
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Article
25-Hydroxycholesterol Effect on Membrane Structure and Mechanical Properties
Int. J. Mol. Sci. 2021, 22(5), 2574; https://doi.org/10.3390/ijms22052574 - 04 Mar 2021
Cited by 2 | Viewed by 597
Abstract
Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses’ entry [...] Read more.
Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses’ entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations. Full article
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Article
SAAFEC-SEQ: A Sequence-Based Method for Predicting the Effect of Single Point Mutations on Protein Thermodynamic Stability
Int. J. Mol. Sci. 2021, 22(2), 606; https://doi.org/10.3390/ijms22020606 - 09 Jan 2021
Cited by 5 | Viewed by 823
Abstract
Modeling the effect of mutations on protein thermodynamics stability is useful for protein engineering and understanding molecular mechanisms of disease-causing variants. Here, we report a new development of the SAAFEC method, the SAAFEC-SEQ, which is a gradient boosting decision tree machine learning method [...] Read more.
Modeling the effect of mutations on protein thermodynamics stability is useful for protein engineering and understanding molecular mechanisms of disease-causing variants. Here, we report a new development of the SAAFEC method, the SAAFEC-SEQ, which is a gradient boosting decision tree machine learning method to predict the change of the folding free energy caused by amino acid substitutions. The method does not require the 3D structure of the corresponding protein, but only its sequence and, thus, can be applied on genome-scale investigations where structural information is very sparse. SAAFEC-SEQ uses physicochemical properties, sequence features, and evolutionary information features to make the predictions. It is shown to consistently outperform all existing state-of-the-art sequence-based methods in both the Pearson correlation coefficient and root-mean-squared-error parameters as benchmarked on several independent datasets. The SAAFEC-SEQ has been implemented into a web server and is available as stand-alone code that can be downloaded and embedded into other researchers’ code. Full article
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2020

Jump to: 2021, 2019

Article
BION-2: Predicting Positions of Non-Specifically Bound Ions on Protein Surface by a Gaussian-Based Treatment of Electrostatics
Int. J. Mol. Sci. 2021, 22(1), 272; https://doi.org/10.3390/ijms22010272 - 29 Dec 2020
Cited by 2 | Viewed by 479
Abstract
Ions play significant roles in biological processes—they may specifically bind to a protein site or bind non-specifically on its surface. Although the role of specifically bound ions ranges from actively providing structural compactness via coordination of charge–charge interactions to numerous enzymatic activities, non-specifically [...] Read more.
Ions play significant roles in biological processes—they may specifically bind to a protein site or bind non-specifically on its surface. Although the role of specifically bound ions ranges from actively providing structural compactness via coordination of charge–charge interactions to numerous enzymatic activities, non-specifically surface-bound ions are also crucial to maintaining a protein’s stability, responding to pH and ion concentration changes, and contributing to other biological processes. However, the experimental determination of the positions of non-specifically bound ions is not trivial, since they may have a low residential time and experience significant thermal fluctuation of their positions. Here, we report a new release of a computational method, the BION-2 method, that predicts the positions of non-specifically surface-bound ions. The BION-2 utilizes the Gaussian-based treatment of ions within the framework of the modified Poisson–Boltzmann equation, which does not require a sharp boundary between the protein and water phase. Thus, the predictions are done by the balance of the energy of interaction between the protein charges and the corresponding ions and the de-solvation penalty of the ions as they approach the protein. The BION-2 is tested against experimentally determined ion’s positions and it is demonstrated that it outperforms the old BION and other available tools. Full article
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Article
Polar Lipid Fraction E from Sulfolobus acidocaldarius and Dipalmitoylphosphatidylcholine Can Form Stable yet Thermo-Sensitive Tetraether/Diester Hybrid Archaeosomes with Controlled Release Capability
Int. J. Mol. Sci. 2020, 21(21), 8388; https://doi.org/10.3390/ijms21218388 - 09 Nov 2020
Cited by 1 | Viewed by 668
Abstract
Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. [...] Read more.
Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. Our present study tackles this long-standing problem. We made hybrid archaeosomes composed of tetraether lipids, called the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius, and the synthetic diester lipid dipalmitoylphosphatidylcholine (DPPC). Differential polarized phase-modulation and steady-state fluorometry, confocal fluorescence microscopy, zeta potential (ZP) measurements, and biochemical assays were employed to characterize the physical properties and drug behaviors in PLFE/DPPC hybrid archaeosomes in the presence and absence of live cells. We found that PLFE lipids have an ordering effect on fluid DPPC liposomal membranes, which can slow down the release of entrapped drugs, while PLFE provides high negative charges on the outer surface of liposomes, which can increase vesicle stability against coalescence among liposomes or with cells. Furthermore, we found that the zeta potential in hybrid archaeosomes with 30 mol% PLFE and 70 mol% DPPC (designated as PLFE/DPPC(3:7) archaeosomes) undergoes an abrupt increase from −48 mV at 37 °C to −16 mV at 44 °C (termed the ZP transition), which we hypothesize results from DPPC domain melting and PLFE lipid ‘flip-flop’. The anticancer drug doxorubicin (DXO) can be readily incorporated into PLFE/DPPC(3:7) archaeosomes. The rate constant of DXO release from PLFE/DPPC(3:7) archaeosomes into Tris buffer exhibited a sharp increase (~2.5 times), when the temperature was raised from 37 to 42 °C, which is believed to result from the liposomal structural changes associated with the ZP transition. This thermo-induced sharp increase in drug release was not affected by serum proteins as a similar temperature dependence of drug release kinetics was observed in human blood serum. A 15-min pre-incubation of PLFE/DPPC(3:7) archaeosomal DXO with MCF-7 breast cancer cells at 42 °C caused a significant increase in the amount of DXO entering into the nuclei and a considerable increase in the cell’s cytotoxicity under the 37 °C growth temperature. Taken together, our data suggests that PLFE/DPPC(3:7) archaeosomes are stable yet potentially useful thermo-sensitive liposomes wherein the temperature range (from 37 to 42–44 °C) clinically used for mild hyperthermia treatment of tumors can be used to trigger drug release for medical interventions. Full article
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Article
Coevolution, Dynamics and Allostery Conspire in Shaping Cooperative Binding and Signal Transmission of the SARS-CoV-2 Spike Protein with Human Angiotensin-Converting Enzyme 2
Int. J. Mol. Sci. 2020, 21(21), 8268; https://doi.org/10.3390/ijms21218268 - 04 Nov 2020
Cited by 6 | Viewed by 981
Abstract
Binding to the host receptor is a critical initial step for the coronavirus SARS-CoV-2 spike protein to enter into target cells and trigger virus transmission. A detailed dynamic and energetic view of the binding mechanisms underlying virus entry is not fully understood and [...] Read more.
Binding to the host receptor is a critical initial step for the coronavirus SARS-CoV-2 spike protein to enter into target cells and trigger virus transmission. A detailed dynamic and energetic view of the binding mechanisms underlying virus entry is not fully understood and the consensus around the molecular origins behind binding preferences of SARS-CoV-2 for binding with the angiotensin-converting enzyme 2 (ACE2) host receptor is yet to be established. In this work, we performed a comprehensive computational investigation in which sequence analysis and modeling of coevolutionary networks are combined with atomistic molecular simulations and comparative binding free energy analysis of the SARS-CoV and SARS-CoV-2 spike protein receptor binding domains with the ACE2 host receptor. Different from other computational studies, we systematically examine the molecular and energetic determinants of the binding mechanisms between SARS-CoV-2 and ACE2 proteins through the lens of coevolution, conformational dynamics, and allosteric interactions that conspire to drive binding interactions and signal transmission. Conformational dynamics analysis revealed the important differences in mobility of the binding interfaces for the SARS-CoV-2 spike protein that are not confined to several binding hotspots, but instead are broadly distributed across many interface residues. Through coevolutionary network analysis and dynamics-based alanine scanning, we established linkages between the binding energy hotspots and potential regulators and carriers of signal communication in the virus–host receptor complexes. The results of this study detailed a binding mechanism in which the energetics of the SARS-CoV-2 association with ACE2 may be determined by cumulative changes of a number of residues distributed across the entire binding interface. The central findings of this study are consistent with structural and biochemical data and highlight drug discovery challenges of inhibiting large and adaptive protein–protein interfaces responsible for virus entry and infection transmission. Full article
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Article
Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores
Int. J. Mol. Sci. 2020, 21(19), 7099; https://doi.org/10.3390/ijms21197099 - 26 Sep 2020
Cited by 3 | Viewed by 776
Abstract
Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) [...] Read more.
Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1–2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment. Full article
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Zooming into the Dark Side of Human Annexin-S100 Complexes: Dynamic Alliance of Flexible Partners
Int. J. Mol. Sci. 2020, 21(16), 5879; https://doi.org/10.3390/ijms21165879 - 16 Aug 2020
Cited by 3 | Viewed by 809
Abstract
Annexins and S100 proteins form two large families of Ca2+-binding proteins. They are quite different both structurally and functionally, with S100 proteins being small (10–12 kDa) acidic regulatory proteins from the EF-hand superfamily of Ca2+-binding proteins, and with annexins [...] Read more.
Annexins and S100 proteins form two large families of Ca2+-binding proteins. They are quite different both structurally and functionally, with S100 proteins being small (10–12 kDa) acidic regulatory proteins from the EF-hand superfamily of Ca2+-binding proteins, and with annexins being at least three-fold larger (329 ± 12 versus 98 ± 7 residues) and using non-EF-hand-based mechanism for calcium binding. Members of both families have multiple biological roles, being able to bind to a large cohort of partners and possessing a multitude of functions. Furthermore, annexins and S100 proteins can interact with each other in either a Ca2+-dependent or Ca2+-independent manner, forming functional annexin-S100 complexes. Such functional polymorphism and binding indiscrimination are rather unexpected, since structural information is available for many annexins and S100 proteins, which therefore are considered as ordered proteins that should follow the classical “one protein–one structure–one function” model. On the other hand, the ability to be engaged in a wide range of interactions with multiple, often unrelated, binding partners and possess multiple functions represent characteristic features of intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs); i.e., functional proteins or protein regions lacking unique tertiary structures. The aim of this paper is to provide an overview of the functional roles of human annexins and S100 proteins, and to use the protein intrinsic disorder perspective to explain their exceptional multifunctionality and binding promiscuity. Full article
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Article
Insights into the Effect of Curcumin and (–)-Epigallocatechin-3-Gallate on the Aggregation of Aβ(1–40) Monomers by Means of Molecular Dynamics
Int. J. Mol. Sci. 2020, 21(15), 5462; https://doi.org/10.3390/ijms21155462 - 30 Jul 2020
Cited by 5 | Viewed by 724
Abstract
In this study, we compared the effects of two well-known natural compounds on the early step of the fibrillation process of amyloid-β (1–40), responsible for the formation of plaques in the brains of patients affected by Alzheimer’s disease (AD). The use of extensive [...] Read more.
In this study, we compared the effects of two well-known natural compounds on the early step of the fibrillation process of amyloid-β (1–40), responsible for the formation of plaques in the brains of patients affected by Alzheimer’s disease (AD). The use of extensive replica exchange simulations up to the µs scale allowed us to characterize the inhibition activity of (–)-epigallocatechin-3-gallate (EGCG) and curcumin (CUR) on unfolded amyloid fibrils. A reduced number of β-strands, characteristic of amyloid fibrils, and an increased distance between the amino acids that are responsible for the intra- and interprotein aggregations are observed. The central core region of the amyloid-β (Aβ(1–40)) fibril is found to have a high affinity to EGCG and CUR due to the presence of hydrophobic residues. Lastly, the free binding energy computed using the Poisson Boltzmann Surface Ares suggests that EGCG is more likely to bind to unfolded Aβ(1–40) fibrils and that this molecule can be a good candidate to develop new and more effective congeners to treat AD. Full article
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Article
Low-Dose Ionizing Radiation Modulates Microglia Phenotypes in the Models of Alzheimer’s Disease
Int. J. Mol. Sci. 2020, 21(12), 4532; https://doi.org/10.3390/ijms21124532 - 25 Jun 2020
Cited by 8 | Viewed by 1840
Abstract
Alzheimer’s disease (AD) is the most common type of dementia. AD involves major pathologies such as amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain. During the progression of AD, microglia can be polarized from anti-inflammatory M2 to pro-inflammatory M1 phenotype. The activation [...] Read more.
Alzheimer’s disease (AD) is the most common type of dementia. AD involves major pathologies such as amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain. During the progression of AD, microglia can be polarized from anti-inflammatory M2 to pro-inflammatory M1 phenotype. The activation of triggering receptor expressed on myeloid cells 2 (TREM2) may result in microglia phenotype switching from M1 to M2, which finally attenuated Aβ deposition and memory loss in AD. Low-dose ionizing radiation (LDIR) is known to ameliorate Aβ pathology and cognitive deficits in AD; however, the therapeutic mechanisms of LDIR against AD-related pathology have been little studied. First, we reconfirm that LDIR (two Gy per fraction for five times)-treated six-month 5XFAD mice exhibited (1) the reduction of Aβ deposition, as reflected by thioflavins S staining, and (2) the improvement of cognitive deficits, as revealed by Morris water maze test, compared to sham-exposed 5XFAD mice. To elucidate the mechanisms of LDIR-induced inhibition of Aβ accumulation and memory loss in AD, we examined whether LDIR regulates the microglial phenotype through the examination of levels of M1 and M2 cytokines in 5XFAD mice. In addition, we investigated the direct effects of LDIR on lipopolysaccharide (LPS)-induced production and secretion of M1/M2 cytokines in the BV-2 microglial cells. In the LPS- and LDIR-treated BV-2 cells, the M2 phenotypic marker CD206 was significantly increased, compared with LPS- and sham-treated BV-2 cells. Finally, the effect of LDIR on M2 polarization was confirmed by detection of increased expression of TREM2 in LPS-induced BV2 cells. These results suggest that LDIR directly induced phenotype switching from M1 to M2 in the brain with AD. Taken together, our results indicated that LDIR modulates LPS- and Aβ-induced neuroinflammation by promoting M2 polarization via TREM2 expression, and has beneficial effects in the AD-related pathology such as Aβ deposition and memory loss. Full article
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Article
Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4
Int. J. Mol. Sci. 2020, 21(12), 4323; https://doi.org/10.3390/ijms21124323 - 17 Jun 2020
Viewed by 1087
Abstract
Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between [...] Read more.
Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators—calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein–protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions. Full article
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Article
Hydrophobic Amino Acids as Universal Elements of Protein-Induced DNA Structure Deformation
Int. J. Mol. Sci. 2020, 21(11), 3986; https://doi.org/10.3390/ijms21113986 - 02 Jun 2020
Cited by 1 | Viewed by 952
Abstract
Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein–DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions [...] Read more.
Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein–DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions with hydrophobic amino acid residues is a universal element of protein–DNA recognition. We provide evidence to support this by associating each DNA minor groove-binding amino acid residue with the local dimensions of the DNA double helix using a novel algorithm. The widened DNA minor grooves are associated with high GC content. However, some AT-rich sequences contacted by hydrophobic amino acids (e.g., phenylalanine) display extreme values of minor groove width as well. For a number of hydrophobic amino acids, distinct secondary structure preferences could be identified for residues interacting with the widened DNA minor groove. These results hold even after discarding the most populous families of minor groove-binding proteins. Full article
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Article
Intrinsic Disorder in Tetratricopeptide Repeat Proteins
Int. J. Mol. Sci. 2020, 21(10), 3709; https://doi.org/10.3390/ijms21103709 - 25 May 2020
Cited by 1 | Viewed by 1080
Abstract
Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of [...] Read more.
Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of TPR domains is their ability to stack continuously one upon the other, with these stacked repeats being able to affect interaction with binding partners either sequentially or in combination. It is known that many repeat-containing proteins are characterized by high levels of intrinsic disorder, and that many protein tandem repeats can be intrinsically disordered. Furthermore, it seems that TPR-containing proteins share many characteristics with hybrid proteins containing ordered domains and intrinsically disordered protein regions. However, there has not been a systematic analysis of the intrinsic disorder status of TPR proteins. To fill this gap, we analyzed 166 human TPR proteins to determine the degree to which proteins containing TPR motifs are affected by intrinsic disorder. Our analysis revealed that these proteins are characterized by different levels of intrinsic disorder and contain functional disordered regions that are utilized for protein-protein interactions and often serve as targets of various posttranslational modifications. Full article
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Article
Characteristic Analysis of Homo- and Heterodimeric Complexes of Human Mitochondrial Pyruvate Carrier Related to Metabolic Diseases
Int. J. Mol. Sci. 2020, 21(9), 3403; https://doi.org/10.3390/ijms21093403 - 11 May 2020
Cited by 3 | Viewed by 1501
Abstract
Human mitochondrial pyruvate carriers (hMPCs), which are required for the uptake of pyruvate into mitochondria, are associated with several metabolic diseases, including type 2 diabetes and various cancers. Yeast MPC was recently demonstrated to form a functional unit of heterodimers. However, human MPC-1 [...] Read more.
Human mitochondrial pyruvate carriers (hMPCs), which are required for the uptake of pyruvate into mitochondria, are associated with several metabolic diseases, including type 2 diabetes and various cancers. Yeast MPC was recently demonstrated to form a functional unit of heterodimers. However, human MPC-1 (hMPC-1) and MPC-2 (hMPC-2) have not yet been individually isolated for their detailed characterization, in particular in terms of their structural and functional properties, namely, whether they exist as homo- or heterodimers. In this study, hMPC-1 and hMPC-2 were successfully isolated in micelles and they formed stable homodimers. However, the heterodimer state was found to be dominant when both hMPC-1 and hMPC-2 were present. In addition, as heterodimers, the molecules exhibited a higher binding capacity to both substrates and inhibitors, together with a larger structural stability than when they existed as homodimers. Taken together, our results demonstrated that the hetero-dimerization of hMPCs is the main functional unit of the pyruvate metabolism, providing a structural insight into the transport mechanisms of hMPCs. Full article
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Article
Data-Driven Modeling Identifies TIRAP-Independent MyD88 Activation Complex and Myddosome Assembly Strategy in LPS/TLR4 Signaling
Int. J. Mol. Sci. 2020, 21(9), 3061; https://doi.org/10.3390/ijms21093061 - 26 Apr 2020
Cited by 11 | Viewed by 1259
Abstract
TLR4 complexes are essential for the initiation of the LPS-induced innate immune response. The Myddosome, which mainly contains TLR4, TIRAP, MyD88, IRAK1/4 and TRAF6 proteins, is regarded as a major complex of TLR4. Although the Myddosome has been well studied, a quantitative description [...] Read more.
TLR4 complexes are essential for the initiation of the LPS-induced innate immune response. The Myddosome, which mainly contains TLR4, TIRAP, MyD88, IRAK1/4 and TRAF6 proteins, is regarded as a major complex of TLR4. Although the Myddosome has been well studied, a quantitative description of the Myddosome assembly dynamics is still lacking. Furthermore, whether some unknown TLR4 complexes exist remains unclear. In this study, we constructed a SWATH-MS data-based mathematical model that describes the component assembly dynamics of TLR4 complexes. In addition to Myddosome, we suggest that a TIRAP-independent MyD88 activation complex is formed upon LPS stimulation, in which TRAF6 is not included. Furthermore, quantitative analysis reveals that the distribution of components in TIRAP-dependent and -independent MyD88 activation complexes are LPS stimulation-dependent. The two complexes compete for recruiting IRAK1/4 proteins. MyD88 forms higher-order assembly in the Myddosome and we show that the strategy to form higher-order assembly is also LPS stimulation-dependent. MyD88 forms a long chain upon weak stimulation, but forms a short chain upon strong stimulation. Higher-order assembly of MyD88 is directly determined by the level of TIRAP in the Myddosome, providing a formation mechanism for efficient signaling transduction. Taken together, our study provides an enhanced understanding of component assembly dynamics and strategies in TLR4 complexes. Full article
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Article
SAAMBE-3D: Predicting Effect of Mutations on Protein–Protein Interactions
Int. J. Mol. Sci. 2020, 21(7), 2563; https://doi.org/10.3390/ijms21072563 - 07 Apr 2020
Cited by 14 | Viewed by 1927
Abstract
Maintaining wild type protein–protein interactions is essential for the normal function of cell and any mutation that alter their characteristics can cause disease. Therefore, the ability to correctly and quickly predict the effect of amino acid mutations is crucial for understanding disease effects [...] Read more.
Maintaining wild type protein–protein interactions is essential for the normal function of cell and any mutation that alter their characteristics can cause disease. Therefore, the ability to correctly and quickly predict the effect of amino acid mutations is crucial for understanding disease effects and to be able to carry out genome-wide studies. Here, we report a new development of the SAAMBE method, SAAMBE-3D, which is a machine learning-based approach, resulting in accurate predictions and is extremely fast. It achieves the Pearson correlation coefficient ranging from 0.78 to 0.82 depending on the training protocol in benchmarking five-fold validation test against the SKEMPI v2.0 database and outperforms currently existing algorithms on various blind-tests. Furthermore, optimized and tested via five-fold cross-validation on the Cornell University dataset, the SAAMBE-3D achieves AUC of 1.0 and 0.96 on a homo and hereto-dimer test datasets. Another important feature of SAAMBE-3D is that it is very fast, it takes less than a fraction of a second to complete a prediction. SAAMBE-3D is available as a web server and as well as a stand-alone code, the last one being another important feature allowing other researchers to directly download the code and run it on their local computer. Combined all together, SAAMBE-3D is an accurate and fast software applicable for genome-wide studies to assess the effect of amino acid mutations on protein–protein interactions. The webserver and the stand-alone codes (SAAMBE-3D for predicting the change of binding free energy and SAAMBE-3D-DN for predicting if the mutation is disruptive or non-disruptive) are available. Full article
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Article
Distal Unfolding of Ferricytochrome c Induced by the F82K Mutation
Int. J. Mol. Sci. 2020, 21(6), 2134; https://doi.org/10.3390/ijms21062134 - 20 Mar 2020
Viewed by 683
Abstract
It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side [...] Read more.
It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82—conserved amino acid and a key actor in cytochrome c intermolecular interactions—with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by 1H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization. Full article
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Article
In Silico Insights into Protein–Protein Interaction Disruptive Mutations in the PCSK9-LDLR Complex
Int. J. Mol. Sci. 2020, 21(5), 1550; https://doi.org/10.3390/ijms21051550 - 25 Feb 2020
Cited by 8 | Viewed by 1543
Abstract
Gain-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to reduced uptake of LDL (low density lipoprotein) cholesterol and, therefore, increased plasma LDL levels. However, the mechanism by which these mutants reduce LDL reuptake is not fully understood. Here, we have used [...] Read more.
Gain-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to reduced uptake of LDL (low density lipoprotein) cholesterol and, therefore, increased plasma LDL levels. However, the mechanism by which these mutants reduce LDL reuptake is not fully understood. Here, we have used molecular dynamics simulations, MM/PBSA (Molecular Mechanics/Poisson–Boltzmann Surface Area) binding affinity calculations, and residue interaction networks, to investigate the protein–protein interaction (PPI) disruptive effects of two of PCSK9′s gain-of-function mutations, Ser127Arg and Asp374Tyr on the PCSK9 and LDL receptor complex. In addition to these PPI disruptive mutants, a third, non-interface mutation (Arg496Trp) is included as a positive control. Our results indicate that Ser127Arg and Asp374Tyr confer significantly improved binding affinity, as well as different binding modes, when compared to the wild-type. These PPI disruptive mutations lie between the EGF(A) (epidermal growth factor precursor homology domain A) of the LDL receptor and the catalytic domain of PCSK9 (Asp374Tyr) and between the prodomain of PCSK9 and the β-propeller of the LDL receptor (Ser127Arg). The interactions involved in these two interfaces result in an LDL receptor that is sterically inhibited from entering its closed conformation. This could potentially implicate the prodomain as a target for small molecule inhibitors. Full article
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Article
Mathematical Model of ATM Activation and Chromatin Relaxation by Ionizing Radiation
Int. J. Mol. Sci. 2020, 21(4), 1214; https://doi.org/10.3390/ijms21041214 - 12 Feb 2020
Cited by 1 | Viewed by 1110
Abstract
We propose a comprehensive mathematical model to study the dynamics of ionizing radiation induced Ataxia-telangiectasia mutated (ATM) activation that consists of ATM activation through dual mechanisms: the initiative activation pathway triggered by the DNA damage-induced local chromatin relaxation and the primary activation pathway [...] Read more.
We propose a comprehensive mathematical model to study the dynamics of ionizing radiation induced Ataxia-telangiectasia mutated (ATM) activation that consists of ATM activation through dual mechanisms: the initiative activation pathway triggered by the DNA damage-induced local chromatin relaxation and the primary activation pathway consisting of a self-activation loop by interplay with chromatin relaxation. The model is expressed as a series of biochemical reactions, governed by a system of differential equations and analyzed by dynamical systems techniques. Radiation induced double strand breaks (DSBs) cause rapid local chromatin relaxation, which is independent of ATM but initiates ATM activation at damage sites. Key to the model description is how chromatin relaxation follows when active ATM phosphorylates KAP-1, which subsequently spreads throughout the chromatin and induces global chromatin relaxation. Additionally, the model describes how oxidative stress activation of ATM triggers a self-activation loop in which PP2A and ATF2 are released so that ATM can undergo autophosphorylation and acetylation for full activation in relaxed chromatin. In contrast, oxidative stress alone can partially activate ATM because phosphorylated ATM remains as a dimer. The model leads to predictions on ATM mediated responses to DSBs, oxidative stress, or both that can be tested by experiments. Full article
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Article
Using the Gibbs Function as a Measure of Human Brain Development Trends from Fetal Stage to Advanced Age
Int. J. Mol. Sci. 2020, 21(3), 1116; https://doi.org/10.3390/ijms21031116 - 07 Feb 2020
Cited by 2 | Viewed by 972
Abstract
We propose to use a Gibbs free energy function as a measure of the human brain development. We adopt this approach to the development of the human brain over the human lifespan: from a prenatal stage to advanced age. We used proteomic expression [...] Read more.
We propose to use a Gibbs free energy function as a measure of the human brain development. We adopt this approach to the development of the human brain over the human lifespan: from a prenatal stage to advanced age. We used proteomic expression data with the Gibbs free energy to quantify human brain’s protein–protein interaction networks. The data, obtained from BioGRID, comprised tissue samples from the 16 main brain areas, at different ages, of 57 post-mortem human brains. We found a consistent functional dependence of the Gibbs free energies on age for most of the areas and both sexes. A significant upward trend in the Gibbs function was found during the fetal stages, which is followed by a sharp drop at birth with a subsequent period of relative stability and a final upward trend toward advanced age. We interpret these data in terms of structure formation followed by its stabilization and eventual deterioration. Furthermore, gender data analysis has uncovered the existence of functional differences, showing male Gibbs function values lower than female at prenatal and neonatal ages, which become higher at ages 8 to 40 and finally converging at late adulthood with the corresponding female Gibbs functions. Full article
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Article
C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W)
Int. J. Mol. Sci. 2020, 21(3), 926; https://doi.org/10.3390/ijms21030926 - 31 Jan 2020
Cited by 4 | Viewed by 1040
Abstract
The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic β cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to [...] Read more.
The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic β cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to tryptophan at position 325 in the C-terminal domain (CTD) increases the risk of developing type 2 diabetes mellitus (T2D). In the present study, the CTDs of hZnT8 (the wild-type (WT) and its disease risk variant (R325W)) were expressed, purified, and characterized in their native forms by biophysical techniques. The data reveal that the CTDs form tetramers which are stabilized by zinc binding, and exhibit negligible differences in their secondary structure content and zinc-binding affinities in solution. These findings provide the basis for conducting further structural studies aimed at unravelling the molecular mechanism underlying the increased susceptibility to develop T2D, which is modulated by the disease risk variant. Full article
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Review
Integrated Computational Approaches and Tools for Allosteric Drug Discovery
Int. J. Mol. Sci. 2020, 21(3), 847; https://doi.org/10.3390/ijms21030847 - 28 Jan 2020
Cited by 20 | Viewed by 2569
Abstract
Understanding molecular mechanisms underlying the complexity of allosteric regulation in proteins has attracted considerable attention in drug discovery due to the benefits and versatility of allosteric modulators in providing desirable selectivity against protein targets while minimizing toxicity and other side effects. The proliferation [...] Read more.
Understanding molecular mechanisms underlying the complexity of allosteric regulation in proteins has attracted considerable attention in drug discovery due to the benefits and versatility of allosteric modulators in providing desirable selectivity against protein targets while minimizing toxicity and other side effects. The proliferation of novel computational approaches for predicting ligand–protein interactions and binding using dynamic and network-centric perspectives has led to new insights into allosteric mechanisms and facilitated computer-based discovery of allosteric drugs. Although no absolute method of experimental and in silico allosteric drug/site discovery exists, current methods are still being improved. As such, the critical analysis and integration of established approaches into robust, reproducible, and customizable computational pipelines with experimental feedback could make allosteric drug discovery more efficient and reliable. In this article, we review computational approaches for allosteric drug discovery and discuss how these tools can be utilized to develop consensus workflows for in silico identification of allosteric sites and modulators with some applications to pathogen resistance and precision medicine. The emerging realization that allosteric modulators can exploit distinct regulatory mechanisms and can provide access to targeted modulation of protein activities could open opportunities for probing biological processes and in silico design of drug combinations with improved therapeutic indices and a broad range of activities. Full article
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Communication
Unveiling the Molecular Basis of the Noonan Syndrome-Causing Mutation T42A of SHP2
Int. J. Mol. Sci. 2020, 21(2), 461; https://doi.org/10.3390/ijms21020461 - 10 Jan 2020
Cited by 1 | Viewed by 1083
Abstract
Noonan syndrome (NS) is a genetic disorder caused by the hyperactivation of the RAS-MAPK molecular pathway. About 50% of NS cases are caused by mutations affecting the SHP2 protein, a multi-domain phosphatase with a fundamental role in the regulation of the RAS-MAPK pathway. [...] Read more.
Noonan syndrome (NS) is a genetic disorder caused by the hyperactivation of the RAS-MAPK molecular pathway. About 50% of NS cases are caused by mutations affecting the SHP2 protein, a multi-domain phosphatase with a fundamental role in the regulation of the RAS-MAPK pathway. Most NS-causing mutations influence the stability of the inactive form of SHP2. However, one NS-causing mutation, namely T42A, occurs in the binding pocket of the N-SH2 domain of the protein. Here, we present a quantitative characterization of the effect of the T42A mutation on the binding of the N-terminal SH2 domain of SHP2 with a peptide mimicking Gab2, a fundamental interaction that triggers the activation of the phosphatase in the cellular environment. Our results show that whilst the T42A mutation does not affect the association rate constant with the ligand, it causes a dramatic increase of the affinity for Gab2. This effect is due to a remarkable decrease of the microscopic dissociation rate constant of over two orders of magnitudes. In an effort to investigate the molecular basis of the T42A mutation in causing Noonan syndrome, we also compare the experimental results with a more conservative variant, T42S. Our findings are discussed in the context of the structural data available on SHP2. Full article
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Article
Conformational Plasticity of the Active Site Entrance in E. coli Aspartate Transcarbamoylase and Its Implication in Feedback Regulation
Int. J. Mol. Sci. 2020, 21(1), 320; https://doi.org/10.3390/ijms21010320 - 03 Jan 2020
Cited by 2 | Viewed by 811
Abstract
Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a “textbook example” for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its [...] Read more.
Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a “textbook example” for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its remote feedback regulation. Herein, we determined a structure of E. coli ATCase in which a key residue located (Arg167) at the entrance of the active site adopted an uncommon open conformation, representing the first wild-type apo-form E. coli ATCase holoenzyme that features this state. Based on the structure and our results of enzymatic characterization, as well as molecular dynamic simulations, we provide new insights into the feedback regulation of E. coli ATCase. We speculate that the binding of pyrimidines or purines would affect the hydrogen bond network at the interface of the catalytic and regulatory subunit, which would further influence the stability of the open conformation of Arg167 and the enzymatic activity of ATCase. Our results not only revealed the importance of the previously unappreciated open conformation of Arg167 in the active site, but also helped to provide rationalization for the mechanism of the remote feedback regulation of ATCase. Full article
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Article
In Situ Proteolysis Condition-Induced Crystallization of the XcpVWX Complex in Different Lattices
Int. J. Mol. Sci. 2020, 21(1), 308; https://doi.org/10.3390/ijms21010308 - 02 Jan 2020
Viewed by 947
Abstract
Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the [...] Read more.
Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the use of in situ proteolysis to remove flexible or unstable structures for improving crystallization and crystal quality. In this work; we investigated the effects of in situ proteolysis by chymotrypsin on the crystallization of the XcpVWX complex from the Type II secretion system of Pseudomonas aeruginosa. Different proteolysis conditions were found to result in two distinct lattices in the same crystallization solution. With a shorter chymotrypsin digestion at a lower concentration; the crystals exhibited a P3 hexagonal lattice that accommodates three complex molecules in one asymmetric unit. By contrast; a longer digestion with chymotrypsin of a 10-fold higher concentration facilitated the formation of a compact P212121 orthorhombic lattice with only one complex molecule in each asymmetric unit. The molecules in the hexagonal lattice have shown high atomic displacement parameter values compared with the ones in the orthorhombic lattice. Taken together; our results clearly demonstrate that different proteolysis conditions can result in the generation of distinct lattices in the same crystallization solution; which can be exploited in order to obtain different crystal forms of a better quality Full article
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2019

Jump to: 2021, 2020

Review
Strategies for Molecular Imprinting and the Evolution of MIP Nanoparticles as Plastic Antibodies—Synthesis and Applications
Int. J. Mol. Sci. 2019, 20(24), 6304; https://doi.org/10.3390/ijms20246304 - 13 Dec 2019
Cited by 26 | Viewed by 1816
Abstract
Materials that can mimic the molecular recognition-based functions found in biology are a significant goal for science and technology. Molecular imprinting is a technology that addresses this challenge by providing polymeric materials with antibody-like recognition characteristics. Recently, significant progress has been achieved in [...] Read more.
Materials that can mimic the molecular recognition-based functions found in biology are a significant goal for science and technology. Molecular imprinting is a technology that addresses this challenge by providing polymeric materials with antibody-like recognition characteristics. Recently, significant progress has been achieved in solving many of the practical problems traditionally associated with molecularly imprinted polymers (MIPs), such as difficulties with imprinting of proteins, poor compatibility with aqueous environments, template leakage, and the presence of heterogeneous populations of binding sites in the polymers that contribute to high levels of non-specific binding. This success is closely related to the technology-driven shift in MIP research from traditional bulk polymer formats into the nanomaterial domain. The aim of this article is to throw light on recent developments in this field and to present a critical discussion of the current state of molecular imprinting and its potential in real world applications. Full article
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Article
Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore
Int. J. Mol. Sci. 2019, 20(23), 6067; https://doi.org/10.3390/ijms20236067 - 02 Dec 2019
Cited by 4 | Viewed by 1232
Abstract
Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical “transparency window” of biological tissues. However, the quantum [...] Read more.
Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical “transparency window” of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV. Full article
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Article
Intrinsic Disorder of the BAF Complex: Roles in Chromatin Remodeling and Disease Development
Int. J. Mol. Sci. 2019, 20(21), 5260; https://doi.org/10.3390/ijms20215260 - 23 Oct 2019
Cited by 5 | Viewed by 1628
Abstract
The two-meter-long DNA is compressed into chromatin in the nucleus of every cell, which serves as a significant barrier to transcription. Therefore, for processes such as replication and transcription to occur, the highly compacted chromatin must be relaxed, and the processes required for [...] Read more.
The two-meter-long DNA is compressed into chromatin in the nucleus of every cell, which serves as a significant barrier to transcription. Therefore, for processes such as replication and transcription to occur, the highly compacted chromatin must be relaxed, and the processes required for chromatin reorganization for the aim of replication or transcription are controlled by ATP-dependent nucleosome remodelers. One of the most highly studied remodelers of this kind is the BRG1- or BRM-associated factor complex (BAF complex, also known as SWItch/sucrose non-fermentable (SWI/SNF) complex), which is crucial for the regulation of gene expression and differentiation in eukaryotes. Chromatin remodeling complex BAF is characterized by a highly polymorphic structure, containing from four to 17 subunits encoded by 29 genes. The aim of this paper is to provide an overview of the role of BAF complex in chromatin remodeling and also to use literature mining and a set of computational and bioinformatics tools to analyze structural properties, intrinsic disorder predisposition, and functionalities of its subunits, along with the description of the relations of different BAF complex subunits to the pathogenesis of various human diseases. Full article
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Communication
Multiplexed Competitive Screening of One-Bead-One-Component Combinatorial Libraries Using a ClonePix 2 Colony Sorter
Int. J. Mol. Sci. 2019, 20(20), 5119; https://doi.org/10.3390/ijms20205119 - 16 Oct 2019
Cited by 5 | Viewed by 1160
Abstract
Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies—beyond mere library blocking and competitive screening—to ensure binding selectivity of selected leads. This work [...] Read more.
Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies—beyond mere library blocking and competitive screening—to ensure binding selectivity of selected leads. This work presents a method for multiplexed solid-phase peptide library screening using a ClonePix 2 Colony Picker that integrates (i) orthogonal fluorescent labeling for positive selection against a target protein and negative selection against competitor species with (ii) semi-quantitative tracking of target vs. competitor binding for every library bead. The ClonePix 2 technology enables global at-a-glance evaluation and customization of the parameters for bead selection to ensure high affinity and selectivity of the isolated leads. A case study is presented by screening a peptide library against green-labeled human immunoglobulin G (IgG) and red-labeled host cell proteins (HCPs) using ClonePix 2 to select HCP-binding ligands for flow-through chromatography applications. Using this approach, 79 peptide ligand candidates (6.6% of the total number of ligands screened) were identified as potential HCP-selective ligands, enabling a potential rate of >3,000 library beads screened per hour. Full article
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Article
Novel Genetic Markers for Early Detection of Elevated Breast Cancer Risk in Women
Int. J. Mol. Sci. 2019, 20(19), 4828; https://doi.org/10.3390/ijms20194828 - 28 Sep 2019
Viewed by 1310
Abstract
This study suggests that two newly discovered variants in the MSH2 gene, which codes for a DNA mismatch repair (MMR) protein, can be associated with a high risk of breast cancer. While variants in the MSH2 gene are known to be linked with [...] Read more.
This study suggests that two newly discovered variants in the MSH2 gene, which codes for a DNA mismatch repair (MMR) protein, can be associated with a high risk of breast cancer. While variants in the MSH2 gene are known to be linked with an elevated cancer risk, the MSH2 gene is not a part of the standard kit for testing patients for elevated breast cancer risk. Here we used the results of genetic testing of women diagnosed with breast cancer, but who did not have variants in BRCA1 and BRCA2 genes. Instead, the test identified four variants with unknown significance (VUS) in the MSH2 gene. Here, we carried in silico analysis to develop a classifier that can distinguish pathogenic from benign mutations in MSH2 genes taken from ClinVar. The classifier was then used to classify VUS in MSH2 genes, and two of them, p.Ala272Val and p.Met592Val, were predicted to be pathogenic mutations. These two mutations were found in women with breast cancer who did not have mutations in BRCA1 and BRCA2 genes, and thus they are suggested to be considered as new bio-markers for the early detection of elevated breast cancer risk. However, before this is done, an in vitro validation of mutation pathogenicity is needed and, moreover, the presence of these mutations should be demonstrated in a higher number of patients or in families with breast cancer history. Full article
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Article
Low Energy Shock Wave Therapy Inhibits Inflammatory Molecules and Suppresses Prostatic Pain and Hypersensitivity in a Capsaicin Induced Prostatitis Model in Rats
Int. J. Mol. Sci. 2019, 20(19), 4777; https://doi.org/10.3390/ijms20194777 - 26 Sep 2019
Cited by 7 | Viewed by 1350
Abstract
The effect of low energy shock wave (LESW) therapy on the changes of inflammatory molecules and pain reaction was studied in a capsaicin (10 mM, 0.1 cc) induced prostatitis model in rats. Intraprostatic capsaicin injection induced a pain reaction, including closing of the [...] Read more.
The effect of low energy shock wave (LESW) therapy on the changes of inflammatory molecules and pain reaction was studied in a capsaicin (10 mM, 0.1 cc) induced prostatitis model in rats. Intraprostatic capsaicin injection induced a pain reaction, including closing of the eyes, hypolocomotion, and tactile allodynia, which effects were ameliorated by LESW treatment. LESW therapy (2Hz, energy flux density of 0.12 mJ/mm2) at 200 and 300 shocks significantly decreased capsaicin-induced inflammatory reactions, reflected by a reduction of tissue edema and inflammatory cells, COX-2 and TNF-α stained positive cells, however, the therapeutic effects were not observed at 100 shocks treated group. Capsaicin-induced IL-1β, COX-2, IL-6, caspase-1, and NGF upregulation on day 3 and 7, while NALP1 and TNF-α upregulation was observed on day 7. LESW significantly suppressed the expression of IL-1β, COX-2, caspase-1, NGF on day 3 and IL-1β, TNF-α, COX-2, NALP1, caspase-1, NGF expression on day 7 in a dose-dependent fashion. LESW has no significant effect on IL-6 expression. Intraprostatic capsaicin injection activates inflammatory molecules and induces prostatic pain and hypersensitivity, which effects were suppressed by LESW. These findings might be the potential mechanisms of LESW therapy for nonbacterial prostatitis in humans. Full article
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Article
Robust Sampling of Defective Pathways in Multiple Myeloma
Int. J. Mol. Sci. 2019, 20(19), 4681; https://doi.org/10.3390/ijms20194681 - 21 Sep 2019
Cited by 1 | Viewed by 818
Abstract
We present the analysis of defective pathways in multiple myeloma (MM) using two recently developed sampling algorithms of the biological pathways: The Fisher’s ratio sampler, and the holdout sampler. We performed the retrospective analyses of different gene expression datasets concerning different aspects of [...] Read more.
We present the analysis of defective pathways in multiple myeloma (MM) using two recently developed sampling algorithms of the biological pathways: The Fisher’s ratio sampler, and the holdout sampler. We performed the retrospective analyses of different gene expression datasets concerning different aspects of the disease, such as the existing difference between bone marrow stromal cells in MM and healthy controls (HC), the gene expression profiling of CD34+ cells in MM and HC, the difference between hyperdiploid and non-hyperdiploid myelomas, and the prediction of the chromosome 13 deletion, to provide a deeper insight into the molecular mechanisms involved in the disease. Our analysis has shown the importance of different altered pathways related to glycosylation, infectious disease, immune system response, different aspects of metabolism, DNA repair, protein recycling and regulation of the transcription of genes involved in the differentiation of myeloid cells. The main difference in genetic pathways between hyperdiploid and non-hyperdiploid myelomas are related to infectious disease, immune system response and protein recycling. Our work provides new insights on the genetic pathways involved in this complex disease and proposes novel targets for future therapies. Full article
Article
Cooperative Binding of KaiB to the KaiC Hexamer Ensures Accurate Circadian Clock Oscillation in Cyanobacteria
Int. J. Mol. Sci. 2019, 20(18), 4550; https://doi.org/10.3390/ijms20184550 - 13 Sep 2019
Cited by 3 | Viewed by 1454
Abstract
The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data [...] Read more.
The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data have identified KaiB–KaiC interaction sites; however, KaiB mutations distal from the binding surfaces can impair KaiB–KaiC interaction and the circadian rhythm. Reportedly, KaiB and KaiC exclusively form a complex in a 6:6 stoichiometry, indicating that KaiB–KaiC hexamer binding shows strong positive cooperativity. Here, mutational analysis was used to investigate the functional significance of this cooperative interaction. Results demonstrate that electrostatic complementarity between KaiB protomers promotes their cooperative assembly, which is indispensable for accurate rhythm generation. SasA does not exhibit such electrostatic complementarity and noncooperatively binds to KaiC. Thus, the findings explain KaiB distal mutation effects, providing mechanistic insights into clock protein interplay. Full article
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Article
Cooperativity and Steep Voltage Dependence in a Bacterial Channel
Int. J. Mol. Sci. 2019, 20(18), 4501; https://doi.org/10.3390/ijms20184501 - 11 Sep 2019
Viewed by 1024
Abstract
This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel [...] Read more.
This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named “triplin.” The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles. Full article
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