ABSTRACT
Spike (S) glycoprotein is the largest structural protein of SARS-CoV-2 virus and the main one involved in anchoring of the host receptor ACE2 through the receptor binding domain (RBD). S protein secondary structure is of great interest for shedding light on various aspects, from functionality to pathogenesis, finally to spectral fingerprint for the design of optical biosensors. In this paper, the secondary structure of SARS-CoV-2 S protein and its constituting components, namely RBD, S1 and S2 regions, are investigated at serological pH by measuring their amide I infrared absorption bands through Attenuated Total Reflection Infrared (ATR-IR) spectroscopy. Experimental data in combination with MultiFOLD predictions, Define Secondary Structure of Proteins (DSSP) web server and Gravy value calculations, provide a comprehensive understanding of RBD, S1, S2, and S proteins in terms of their secondary structure content, conformational order, and interaction with the solvent.
ABSTRACT
Cytochrome C (cyt C), the protein involved in oxidative phosphorylation, plays several other crucial roles necessary for both cell life and death. Studying natural variants of cyt C offers the possibility to better characterize the structure-to-function relationship that modulates the different activities of this protein. Naturally mutations in human cyt C (G41S and Y48H) occur in the protein central Ω-loop and cause thrombocytopenia 4. In this study, we have investigated the binding of such variants and of wild type (wt) cyt C to synthetic cardiolipin-containing vesicles. The mutants have a lower propensity in membrane binding, displaying higher dissociation constants with respect to the wt protein. Compressibility measurements reveal that both variants are more flexible than the wt, suggesting that the native central Ω-loop is important for the interaction with membranes. Such hypothesis is supported by molecular dynamics simulations. A minimal distance analysis indicates that in the presence of cardiolipin the central Ω-loop of the mutants is no more in contact with the membrane, as it happens instead in the case of wt cyt C. Such finding might provide a hint for the reduced membrane binding capacity of the variants and their enhanced peroxidase activity in vivo.
Subject(s)
Cardiolipins , Cytochromes c , Molecular Dynamics Simulation , Protein Binding , Cytochromes c/metabolism , Cytochromes c/chemistry , Cytochromes c/genetics , Humans , Cardiolipins/metabolism , Cardiolipins/chemistry , Mutation , Cell Membrane/metabolismABSTRACT
The advent of deep learning algorithms for protein folding opened a new era in the ability of predicting and optimizing the function of proteins once the sequence is known. The task is more intricate when cofactors like metal ions or small ligands are essential to functioning. In this case, the combined use of traditional simulation methods based on interatomic force fields and deep learning predictions is mandatory. We use the example of [FeFe] hydrogenases, enzymes of unicellular algae promising for biotechnology applications to illustrate this situation. [FeFe] hydrogenase is an iron-sulfur protein that catalyzes the chemical reduction of protons dissolved in liquid water into molecular hydrogen as a gas. Hydrogen production efficiency and cell sensitivity to dioxygen are important parameters to optimize the industrial applications of biological hydrogen production. Both parameters are related to the organization of iron-sulfur clusters within protein domains. In this work, we propose possible three-dimensional structures of Chlorella vulgaris 211/11P [FeFe] hydrogenase, the sequence of which was extracted from the recently published genome of the given strain. Initial structural models are built using: (i) the deep learning algorithm AlphaFold; (ii) the homology modeling server SwissModel; (iii) a manual construction based on the best known bacterial crystal structure. Missing iron-sulfur clusters are included and microsecond-long molecular dynamics of initial structures embedded into the water solution environment were performed. Multiple-walkers metadynamics was also used to enhance the sampling of structures encompassing both functional and non-functional organizations of iron-sulfur clusters. The resulting structural model provided by deep learning is consistent with functional [FeFe] hydrogenase characterized by peculiar interactions between cofactors and the protein matrix.
Subject(s)
Chlorella vulgaris , Hydrogenase , Metals , Iron , Hydrogen , Sulfur , WaterABSTRACT
Copper (Cu), with its ability to exist in various oxidation states, notably Cu(I) and Cu(II), plays a crucial role in diverse biological redox reactions. This includes its involvement in pathways associated with oxidative stress in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Transmissible Spongiform Encephalopathies. This paper offers an overview of X-ray Absorption Spectroscopy (XAS) studies designed to elucidate the interactions between Cu ions and proteins or peptides associated with these neurodegenerative diseases. The emphasis lies on XAS specificity, revealing the local coordination environment, and on its sensitivity to Cu oxidation states. Furthermore, the paper focuses on XAS applications targeting the characterization of intermediate reaction states and explores the opportunities arising from recent advancements in time-resolved XAS at ultrabright synchrotron and Free Electron Laser radiation sources.
Subject(s)
Copper , Neurodegenerative Diseases , Oxidation-Reduction , X-Ray Absorption Spectroscopy , Copper/chemistry , Neurodegenerative Diseases/diagnostic imaging , Neurodegenerative Diseases/metabolism , HumansABSTRACT
Lipoxygenases (LOXs) are a family of enzymes that includes different fatty acid oxygenases with a common tridimensional structure. The main functions of LOXs are the production of signaling compounds and the structural modifications of biological membranes. These features of LOXs, their widespread presence in all living organisms, and their involvement in human diseases have attracted the attention of the scientific community over the last decades, leading to several studies mainly focused on understanding their catalytic mechanism and designing effective inhibitors. The aim of this review is to discuss the state-of-the-art of a different, much less explored aspect of LOXs, that is, their interaction with lipid bilayers. To this end, the general architecture of six relevant LOXs (namely human 5-, 12-, and 15-LOX, rabbit 12/15-LOX, coral 8-LOX, and soybean 15-LOX), with different specificity towards the fatty acid substrates, is analyzed through the available crystallographic models. Then, their putative interface with a model membrane is examined in the frame of the conformational flexibility of LOXs, that is due to their peculiar tertiary structure. Finally, the possible future developments that emerge from the available data are discussed.
Subject(s)
Lipid Bilayers , Lipoxygenases , Animals , Humans , Rabbits , Molecular Conformation , Fatty AcidsABSTRACT
Mitogen-activated protein kinases 1 and 3 (MAPK1 and MAPK3), also called extracellular regulated kinases (ERK2 and ERK1), are serine/threonine kinase activated downstream by the Ras/Raf/MEK/ERK signal transduction cascade that regulates a variety of cellular processes. A dysregulation of MAPK cascade is frequently associated to missense mutations on its protein components and may be related to many pathologies, including cancer. In this study we selected from COSMIC database a set of MAPK1 and MAPK3 somatic variants found in cancer tissues carrying missense mutations distributed all over the MAPK1 and MAPK3 sequences. The proteins were expressed as pure recombinant proteins, and their biochemical and biophysical properties have been studied in comparison with the wild type. The missense mutations lead to changes in the tertiary arrangements of all the variants. The thermodynamic stability of the wild type and variants has been investigated in the non-phosphorylated and in the phosphorylated form. Significant differences in the thermal stabilities of most of the variants have been observed, as well as changes in the catalytic efficiencies. The energetics of the catalytic reaction is affected for all the variants for both the MAPK proteins. The stability changes and the variation in the enzyme catalysis observed for most of MAPK1/3 variants suggest that a local change in a residue, distant from the catalytic site, may have long-distance effects that reflect globally on enzyme stability and functions.
Subject(s)
Mutation, Missense , Neoplasms , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mutation, Missense/genetics , Neoplasms/genetics , Neoplasms/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Signal TransductionABSTRACT
Human endo-lysosomes possess a class of proteins called TPC channels on their membrane, which are essential for proper cell functioning. This protein family can be functionally studied by expressing them in plant vacuoles. Inhibition of hTPC activity by naringenin, one of the main flavonoids present in the human diet, has the potential to be beneficial in severe human diseases such as solid tumor development, melanoma, and viral infections. We attempted to identify the molecular basis of the interaction between hTPC2 and naringenin, using ensemble docking on molecular dynamics (MD) trajectories, but the specific binding site remains elusive, posing a challenge that could potentially be addressed in the future by increased computational power in MD and the combined use of microscopy techniques such as cryo-EM.
Subject(s)
Endometriosis , Flavanones , Humans , Female , Flavonoids/pharmacology , Flavonoids/therapeutic use , Flavanones/pharmacology , Flavanones/therapeutic use , Binding SitesABSTRACT
Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed in silico. In particular, we used the available crystallographic structure of a C-terminal fragment of TRAF2 (168 out of 501 a.a.), TRAF2-C, and that of a longer construct, addressed as TRAF2-plus, that we have re-constructed using the AlphaFold2 code. The results indicate that the longer N-terminal tail of TRAF2-plus has a strong influence on the dynamics of the globular regions in the protein C-terminal head. In fact, the quaternary interactions among the TRAF2-C subunits change asymmetrically in time, while the movements of TRAF2-plus monomers are rather limited and more ordered than those of the shorter construct. Such findings shed a new light on the dynamics of TRAF subunits and on the protein mechanism in vivo, since TRAF monomer-trimer equilibrium is crucial for several reasons (receptor recognition, membrane binding, hetero-oligomerization).
Subject(s)
Molecular Dynamics Simulation , Receptors, Tumor Necrosis Factor , TNF Receptor-Associated Factor 2/chemistry , TNF Receptor-Associated Factor 2/metabolism , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction , Ubiquitin-Protein Ligases , NF-kappa B/metabolism , Protein BindingABSTRACT
Amyloid-ß peptide (Aß) aggregates are known to be correlated with pathological neurodegenerative diseases. The fibril formation process of such peptides in solution is influenced by several factors, such as the ionic strength of the buffer, concentration, pH, and presence of other molecules, just to mention a few. In this paper, we report a detailed analysis of in vitro Aß42 fibril formation in the presence of cortisol at different relative concentrations. The thioflavin T fluorescence assay allowed us to monitor the fibril formation kinetics, while a morphological characterization of the aggregates was obtained by atomic force microscopy. Moreover, infrared absorption spectroscopy was exploited to investigate the secondary structure changes along the fibril formation path. Molecular dynamics calculations allowed us to understand the intermolecular interactions with cortisol. The combined results demonstrated the influence of cortisol on the fibril formation process: indeed, at cortisol-Aß42 concentration ratio (ρ) close to 0.1 a faster organization of Aß42 fragments into fibrils is promoted, while for ρ = 1 the formation of fibrils is completely inhibited.
Subject(s)
Amyloid beta-Peptides , Hydrocortisone , Amyloid/chemistry , Amyloid beta-Peptides/chemistry , Kinetics , Peptide Fragments/chemistryABSTRACT
The potential role of styrene oxide in altering the dopaminergic pathway in the ear is investigated by means of molecular docking and molecular dynamics simulations. We estimate the binding affinity of both styrene oxide and dopamine to the dopaminergic receptor DrD2 by computing the free-energy difference, ∆G, between the configuration where the ligand is bound to the receptor and the situation in which it is "infinitely" far away from it. The results show that the styrene oxide has a somewhat lower affinity for binding with respect to dopamine, which, however, may not be enough to prevent exogenous high concentration styrene oxide to compete with endogenous dopamine for DrD2 binding.
Subject(s)
Dopamine , Styrene , Ligands , Molecular Docking Simulation , Protein BindingABSTRACT
A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.
Subject(s)
Ion Channels , Lysosomal Storage Diseases , Humans , Intracellular Membranes/metabolism , Ion Channels/metabolism , Ions/metabolism , Lysosomal Storage Diseases/metabolism , Lysosomes/metabolism , Patch-Clamp TechniquesABSTRACT
Dipole Strength (DS) of the amides has gained a renewed interest in chemical physics since it provides an important tool to disclose the on-site vibrational energy distributions. Apart from earlier experimental efforts on polypeptides, little is still known about DS in complex proteins. We accurately measured the Fourier Transform Infrared absorption spectra of nine proteins in water solution obtaining their Molar Extinction Coefficient in the amide I and II spectral region. Our results show that the amide I DS value depends on the protein secondary structure, being that of the α-rich and unstructured proteins lower by a factor of 2 than that of the ß-rich proteins. The average DS for amino acids in α and ß secondary structures confirms this finding. Normal Mode calculation and Molecular Dynamics were performed and used as tools for data analysis and interpretation. The present outcomes corroborate the hypothesis that antiparallel ß-sheet environment is more prone to delocalize the on-site CO stretching vibration through coupling mechanisms between carbonyl groups, whereas α-helix structures are energetically less stable to permit vibrational mode delocalization.
Subject(s)
Amides/chemistry , Proteins/chemistry , Water/chemistry , Molecular Dynamics Simulation , Protein Structure, Secondary , Spectrophotometry, Infrared , Spectroscopy, Fourier Transform InfraredABSTRACT
TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.
Subject(s)
Protein Subunits/chemistry , TNF Receptor-Associated Factor 2/chemistry , Tyrosine/chemistry , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Inhibitor of Apoptosis Proteins/chemistry , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Proprotein Convertases/chemistry , Proprotein Convertases/genetics , Proprotein Convertases/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Multimerization , Protein Structure, Tertiary , Protein Subunits/genetics , Protein Subunits/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/genetics , TNF Receptor-Associated Death Domain Protein/metabolism , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 2/metabolism , Thermodynamics , Tyrosine/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.
Subject(s)
COVID-19 Drug Treatment , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Flavanones/pharmacology , Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Arabidopsis/metabolism , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , Calcium Channel Blockers/therapeutic use , Drug Evaluation, Preclinical , Endosomes/drug effects , Endosomes/metabolism , Endosomes/virology , Flavanones/therapeutic use , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Lysosomes/virology , Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Pandemics/prevention & control , SARS-CoV-2/pathogenicity , Vacuoles/metabolism , Virus Internalization/drug effectsABSTRACT
The oligomeric state of TRAF2 (tumor necrosis factor-receptor associated factor 2), a TNF (tumor necrosis factor) receptor-associated factor, is crucial for membrane binding and probably plays a fundamental role in regulating the protein function in vivo. In this study we have combined molecular dynamics with the protein contact network approach to characterize the interaction of the three identical subunits of TRAF2. The average structure obtained after a 225 ns simulation reveals that two clusters of different size are formed, one of which includes almost completely two subunits, while the third monomer appears to be more independent. This picture is also confirmed by the estimated average number of inter-subunit contacts and by the comparison of side chains mobility in each monomer. The analysis of equilibrium pressure-induced dissociation measurements supports such findings, indicating that the dimeric-monomeric (2 + 1) might be prevalent with respect to the trimeric configuration, especially in the case of more diluted samples. These findings suggest that the formation of monomeric species, which is crucial for the formation of intra-luminal vesicles, might depend on preferential asymmetric interactions among the three subunits.Communicated by Ramaswamy H. Sarma.
Subject(s)
Molecular Dynamics Simulation , Receptors, Tumor Necrosis Factor , Macromolecular Substances , NF-kappa B/metabolism , Receptors, Tumor Necrosis Factor/metabolism , TNF Receptor-Associated Factor 2/metabolism , Tumor Necrosis FactorsABSTRACT
In the present work we illustrate the results of classical molecular dynamics simulations of model systems composed of six insulin molecules in water in the presence and in the absence of either epigallocatechin-3-gallate or melatonin molecules. For each model system, we performed three independent simulations (replicas) to study the aggregate formation dynamics and insulin interaction with epigallocatechin-3-gallate and melatonin. We find that melatonin is less stably close to insulin with respect to epigallocatechin-3-gallate, which interacts more stably with insulin molecules and mainly with insulin's chain B hydrophobic residues. We observe that the shape of the insulin-aggregated structures in the three model systems is different and depends on whether epigallocatechin-3-gallate is present or not. Simulations show that in the absence of epigallocatechin-3-gallate, insulin molecules tend to form linear aggregates, while in the presence of epigallocatechin-3-gallate, aggregates display a globular shape, less prone to form fibril structures.
Subject(s)
Catechin/analogs & derivatives , Insulin/chemistry , Melatonin/chemistry , Molecular Dynamics Simulation , Catechin/chemistry , Models, Chemical , Models, Molecular , Molecular StructureABSTRACT
Human frataxin is an iron-binding protein involved in the mitochondrial iron-sulfur (Fe-S) clusters assembly, a process fundamental for the functional activity of mitochondrial proteins. Decreased level of frataxin expression is associated with the neurodegenerative disease Friedreich ataxia. Defective function of frataxin may cause defects in mitochondria, leading to increased tumorigenesis. Tumor-initiating cells show higher iron uptake, a decrease in iron storage and a reduced Fe-S clusters synthesis and utilization. In this study, we selected, from COSMIC database, the somatic human frataxin missense variants found in cancer tissues p.D104G, p.A107V, p.F109L, p.Y123S, p.S161I, p.W173C, p.S181F, and p.S202F to analyze the effect of the single amino acid substitutions on frataxin structure, function, and stability. The spectral properties, the thermodynamic and the kinetic stability, as well as the molecular dynamics of the frataxin missense variants found in cancer tissues point to local changes confined to the environment of the mutated residues. The global fold of the variants is not altered by the amino acid substitutions; however, some of the variants show a decreased stability and a decreased functional activity in comparison with that of the wild-type protein.
Subject(s)
Iron-Binding Proteins/chemistry , Iron-Binding Proteins/genetics , Mutation, Missense , Neoplasms/genetics , Amino Acid Substitution , Databases, Genetic , Humans , Models, Molecular , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Conformation , Protein Stability , FrataxinABSTRACT
Genome polymorphisms are responsible for phenotypic differences between humans and for individual susceptibility to genetic diseases and therapeutic responses. Non-synonymous single-nucleotide polymorphisms (nsSNPs) lead to protein variants with a change in the amino acid sequence that may affect the structure and/or function of the protein and may be utilized as efficient structural and functional markers of association to complex diseases. This study is focused on nsSNP variants of the ligand binding domain of PPARγ a nuclear receptor in the superfamily of ligand inducible transcription factors that play an important role in regulating lipid metabolism and in several processes ranging from cellular differentiation and development to carcinogenesis. Here we selected nine nsSNPs variants of the PPARγ ligand binding domain, V290M, R357A, R397C, F360L, P467L, Q286P, R288H, E324K, and E460K, expressed in cancer tissues and/or associated with partial lipodystrophy and insulin resistance. The effects of a single amino acid change on the thermodynamic stability of PPARγ, its spectral properties, and molecular dynamics have been investigated. The nsSNPs PPARγ variants show alteration of dynamics and tertiary contacts that impair the correct reciprocal positioning of helices 3 and 12, crucially important for PPARγ functioning.
Subject(s)
PPAR gamma/chemistry , PPAR gamma/genetics , Polymorphism, Single Nucleotide , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Circular Dichroism , Humans , Ligands , Molecular Dynamics Simulation , PPAR gamma/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Unfolding/drug effects , Structure-Activity Relationship , Thermodynamics , Transcription, Genetic , Urea/pharmacologyABSTRACT
In this work we analyze at a structural level the mechanism by which Cu(II) and Zn(II) ions compete for binding to the Aß peptides that is involved in the etiology of Alzheimer's disease. We collected X-ray absorption spectroscopy data on samples containing Aß with Cu and Zn at different concentration ratios. We show that the order in which metals are added to the peptide solution matters and that, when Zn is added first, it prevents Cu from binding. On the contrary, when Cu is added first, it does not (completely) prevent Zn binding to Aß peptides. Our analysis suggests that Cu and Zn ions are coordinated to different numbers of histidine residues depending on the [ion]:[peptide] concentration ratio.
Subject(s)
Amyloid beta-Peptides/chemistry , Copper/chemistry , X-Ray Absorption Spectroscopy , Zinc/chemistry , Binding SitesABSTRACT
TNF receptor-associated factors (TRAFs) are characterized by an oligomeric structure that plays a fundamental role in the binding process with membrane receptors. In this work, we studied the trimer-to-monomer (T â 3M) equilibrium transition of the TRAF2 C-terminal domain using both chemical (dilution/guanidinium hydrochloride) and mechanical stress (high pressure) to induce the dissociation of the native protein into subunits. The experimental results and computer simulations indicate that stable monomers exist and that their population accounts for 15% of the total TRAF2 molecules already at a physiological intracellular concentration (≈1 µM), being instead the predominant species in the nanomolar concentration range. Because the total amount of TRAF2 changes during a cell cycle, the monomer-trimer equilibrium can be crucial for regulating the activities of TRAF2 in vivo.