Tyr Residues Research Articles

Effects of Pulsed Electric Field on the Physicochemical and Structural Properties of Micellar Casein.

Pulsed electric field (PEF) as a green processing technology is drawing greater attention due to its eco-friendliness and potential to promote sustainable development goals. In this study, the effects of different electric field strengths (EFS, 0-30 kV/cm) on the structure and physicochemical features of casein micelles (CSMs) were investigated. It was found that the particle sizes of CSMs increased at low EFS (10 kV/cm) but decreased at high EFS (30 kV/cm). The absolute ζ-potential at 30 kV/cm increased from -26.6 (native CSMs) to -29.5 mV. Moreover, it was noticed that PEF treatment leads to changes in the surface hydrophobicity; it slightly increased at low EFS (10 kV/cm) but decreased at EFS > 10 kV/cm. PEF enhanced the protein solubility from 84.9 (native CSMs) to 87.1% (at 10 kV/cm). PEF at low EFS (10 kV/cm) intensified the emission fluorescence spectrum of CSMs, while higher EFS reduced the fluorescence intensity compared to native CSMs. Moreover, the analysis of the Amide Ι region showed that PEF-treated CSMs reduced the α-helix and increased the β-sheet content. Raman spectra confirmed that PEF treatment > 10 kV/cm buried tyrosine (Tyr) residues in a hydrophobic environment. It was also found that PEF treatment mainly induced changes in the disulfide linkages. In conclusion, PEF technology can be employed as an eco-friendly technology to change the structure and physiochemical properties of CSMs; this could improve their techno-functional properties.

Peptide Diversification through Addition Reaction of Free Carboxylic Acids to Ynamides

Peptide modification has emerged as an important topic in the academic community and pharmaceutical industry. However, they are primarily focused on the diversification of amines, thiols, and alcohols. Direct and chemoselective modification of acid residues in peptides is relatively underdeveloped. In this context, we report a novel and efficient method for the direct functionalization of acid residues in peptides. By using ynamides as reaction partners, the adducts are rapidly obtained in moderate to excellent yields at room temperature in water. This approach shows excellent chemoselectivity and a broad scope including dipeptides bearing unprotected Trp or Tyr residue and free Ser or Gln residue.

Cytochrome c - silver nanoparticle interactions: Spectroscopy, thermodynamic and enzymatic activity studies

Cytochrome c, an iron containing metalloprotein in the mitochondria of the cells with an oxide/redox property, plays key role in the cell apoptotic pathway. In this study, the interaction of silver nanoparticles (AgNPs) with cytochrome c (Cyt c) was investigated by using a combination of spectroscopic, imaging and thermodynamic techniques, including dynamic light scattering (DLS), ultraviolet–visible (UV–vis) spectroscopy, transmission electron microscopy (TEM), fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). DLS and UV–vis analysis evidenced the formation of surface complexes of Cyt c on AgNPs. The saturation of surface coverage of AgNPs was observed at 4.36 Cyt c molecules per nm2 of AgNPs. The surface complexation resulted in a promotion of the Ag dissolution overtime. The negative sign of enthalpic (ΔH) contribution suggested that electrostatic forces are indicative forces in the interaction between protein and AgNPs. Moreover, the fluorescence spectra revealed that the conformation of protein was altered around tryptophan (Trp) and tyrosine (Tyr) residues indicating the alteration of the tertiary structure of Cyt c. CD analysis evidenced that the secondary structure of Cyt c was modified under AgNPs-Cyt c interactions and the binding of Cyt c onto AgNPs resulted in remarkable structural perturbation around the active site heme, which in turn alter the protein enzymatic activity. The results of the present study contributed to a deeper insight on the mechanisms of interaction between NPs and biomacromolecules and could help establish the in vivo fate of AgNPs on cellular redox homeostasis.

Comprehensive views toward the biomolecular recognition of an anticancer drug, leflunomide with human serum albumin

Biomolecular association of an anticancer drug, leflunomide (LEF) with human serum albumin (HSA), the leading ligands carrier in human circulation was characterized using biophysical (i.e., fluorescence, absorption and voltammetric) methods and computational (i.e., molecular docking and molecular dynamics simulation) techniques. Evaluations of fluorescence, absorption and voltammetric findings endorsed the complex formation between LEF and HSA. An inverse relationship of Stern-Volmer constant–temperature and hyperchromic shift of the protein’s absorption signal with addition of LEF confirmed the LEF quenched the HSA fluorescence through static process. Moderate nature of binding strength (binding constant = 2.76–4.77 × 104 M−1) was detected towards the LEF–HSA complexation, while the association process was naturally driven via hydrophobic interactions, van der Waals interactions and hydrogen bonds, as evident from changes in entropy (ΔS= + 19.91 J mol−1 K−1) and enthalpy (ΔH = − 20.09 kJ mol−1), and molecular docking assessments. Spectral analyses of synchronous and three-dimensional fluorescence validated microenvironmental fluctuations near Trp and Tyr residues upon LEF binding to the protein. LEF association with HSA significantly defended temperature-induced destabilization of the protein. Although LEF was found to attach to HSA at Sudlow’s sites I and II, but exhibited greater preference toward its site I, as detected by the investigations of competitive site-marker displacement. Molecular dynamics simulation assessment revealed that the complex attained equilibrium throughout simulations, showing the LEF–HSA complex constancy. Communicated by Ramaswamy H. Sarma

SARS-CoV-2 inhibitory activity of a short peptide derived from internal fusion peptide of S2 subunit of spike glycoprotein

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has posed a great concern in human population. To fight coronavirus emergence, we have dissected the conserved amino acid region of the internal fusion peptide in the S2 subunit of Spike glycoprotein of SARS-CoV-2 to design new inhibitory peptides. Among the 11 overlapping peptides (9-23-mer), PN19, a 19-mer peptide, exhibited a powerful inhibitory activity against different SARS-CoV-2 clinical isolate variants in absence of cytotoxicity. The PN19 inhibitory activity was found to be dependent on conservation of the central Phe and C-terminal Tyr residues in the peptide sequence. Circular dichroism spectra of the active peptide exhibited an alpha-helix propensity, confirmed by secondary structure prediction analysis. The PN19 inhibitory activity, exerted in the first step of virus infection, was reduced after peptide adsorption treatment with virus-cell substrate during fusion interaction. Additionally, PN19 inhibitory activity was reduced by adding S2 membrane-proximal region derived peptides. PN19 showed binding ability to the S2 membrane proximal region derived peptides, confirmed by molecular modelling, playing a role in the mechanism of action. Collectively, these results confirm that the internal fusion peptide region is a good candidate on which develop peptidomimetic anti SARS-CoV-2 antivirals.

Multi-spectroscopies and molecular simulation insights into the binding of bovine serum albumin and sodium tripolyphosphate

Sodium tripolyphosphate (STPP) is an odorless and water-soluble anionic mineral compound. STPP is used as a separator, texturizer, emulsifier and buffer in the food and detergent industries. Considering the importance of the topic and the interaction of food additives with biological molecules including blood serum albumin, we investigated the binding properties of STPP food additive to blood serum albumin through UV–Vis spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and molecular docking analysis. According to the results, after forming a complex with BSA, STPP reduced the fluorescence intensity of BSA through static quenching mechanisms, and the UV–Vis absorption results were also confirmatory. Synchronous fluorescence and FT-IR results showed that the microenvironment and secondary structure of BSA changed in the presence of STPP. The synchronous fluorescence study has shown that the quenching toward Trp residue is higher than Tyr residue. The thermodynamic results showed that van der Waals force and hydrogen bonding play the main roles in the formation of the STPP-BSA complex. The binding constant decreases from 2.23 × 106 to 4.25 × 101 (M−1) with increasing temperature, indicating a decrease in complex formation due to the interaction of STPP with BSA. Furthermore, molecular docking studies showed that STPP binds to the cavity between domains IIIA and IIIB by van der Waals forces and hydrogen bonds through residues Ser 418, Thr 419, Tyr 496, Lys 499, and Lys 533.

Improving hydrogen peroxide stability of a dye-decolorising peroxidase from Irpex lacteus F17 by site-directed mutagenesis

Il-DyP4, a dye-decolorizing peroxidase (DyP) from Irpex lacteus F17, has a strong ability to oxidise harmful aromatic compounds, implying application potential in the field of environmental protection. However, the low H2O2 stability of Il-DyP4 hinders its practical application. To improve the H2O2 stability of Il-DyP4, oxidation-sensitive amino acids including three Met, one Cys and ten Tyr residues were mutated to less easily oxidizable residues such as Leu, Val and Phe by site-specific mutagenesis. Mutation of the M207 site, located on the surface of the enzyme and far away from the haem group, improved H2O2 stability by 5.3-fold or more. In addition, Y234, also on the enzyme surface, was replaced with Phe and the Y234F mutant displayed 7.3-fold increased H2O2 stability. Furthermore, mutants M207V and Y234F were selected for the decolorization of various types of synthetic dyes, revealing high decolorization activities, especially for M207V with enhanced catalytic activity. The stable DyP mutant M207V has the potential for decolorization of industrial dye wastewater.

Activation of insulin-like growth factor-1 receptor (IGF-1R) promotes growth of colorectal cancer through triggering the MEX3A-mediated degradation of RIG-I.

Insulin-like growth factor-1 receptor (IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers. However, targeting it has often produced the disappointing results as the role played by cross talk with numerous downstream signalings. Here, we report a disobliging IGF-1R signaling which promotes growth of cancer through triggering the E3 ubiquitin ligase MEX3A-mediated degradation of RIG-I. The active β-arrestin-2 scaffolds this disobliging signaling to talk with MEX3A. In response to ligands, IGF-1Rβ activated the basal βarr2 into its active state by phosphorylating the interdomain domain on Tyr64 and Tyr250, opening the middle loop (Leu130‒Cys141) to the RING domain of MEX3A through the conformational changes of βarr2. The models of βarr2/IGF-1Rβ and βarr2/MEX3A could interpret the mechanism of the activated-IGF-1R in triggering degradation of RIG-I. The assay of the mutants βarr2Y64A and βarr2Y250A further confirmed the role of these two Tyr residues of the interlobe in mediating the talk between IGF-1Rβ and the RING domain of MEX3A. The truncated-βarr2 and the peptide ATQAIRIF, which mimicked the RING domain of MEX3A could prevent the formation of βarr2/IGF-1Rβ and βarr2/MEX3A complexes, thus blocking the IGF-1R-triggered RIG-I degradation. Degradation of RIG-I resulted in the suppression of the IFN-I-associated immune cells in the TME due to the blockade of the RIG-I-MAVS-IFN-I pathway. Poly(I:C) could reverse anti-PD-L1 insensitivity by recovery of RIG-I. In summary, we revealed a disobliging IGF-1R signaling by which IGF-1Rβ promoted cancer growth through triggering the MEX3A-mediated degradation of RIG-I.

Structure-Activity Relationship Study of Antimicrobial Peptide PE2 Delivered Novel Linear Derivatives with Potential of Eradicating Biofilms and Low Incidence of Drug Resistance.

The ongoing emergence of antibiotic-resistant pathogens had been dramatically stimulating and accelerating the need for new drugs. PE2 is a kind of cyclic lipopeptide with broad-spectrum antimicrobial activity. Herein, its structure-activity relationship was systematically investigated by employing 4 cyclic analogues and 23 linear analogues for the first time. The screened linear analogues 26 and 27 bearing different fatty acyls at N-termini and a Tyr residue at the 9th position had superior potency compared to the cyclic analogues and showed equivalent antimicrobial activity compared with PE2. Notably, 26 and 27 exhibited significant ability against multidrug-resistant bacteria, favorable resistance to protease, excellent performance against biofilm, low drug resistance, and high effectiveness against the mice pneumonia model. The antibacterial mechanisms of PE2 and linear derivatives 26 and 27 were also preliminarily explored in this study. As described above, 26 and 27 are promising antimicrobial candidates for the treatment of infections associated with drug-resistant bacteria.

The inflammatory oxidant peroxynitrous acid modulates the structure and function of the recombinant human V3 isoform of the extracellular matrix proteoglycan versican

Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO−/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO−/ONOOH. Both reagent ONOO−/ONOOH and SIN-1 (a thermal source of ONOO−/ONOOH) modified Tyr, Trp and Met residues. ONOO−/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr161), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO−/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO−/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.

Exploring the binding behavior mechanism of vitamin B12 to α-Casein and β-Casein: multi-spectroscopy and molecular dynamic approaches

The aim of this study was to investigate the behavior interaction of α-Casein-B12 and β-Casein-B12 complexes as binary systems through the methods of multiple spectroscopic, zeta potential, calorimetric, and molecular dynamics (MD) simulation. Fluorescence spectroscopy denoted the role ofB12as a quencher in both cases of α-Casein and β-Casein fluorescence intensities, which also verifies the existence of interactions. The quenching constants of α-Casein-B12 and β-Casein-B12 complexes at 298 K in the first set of binding sites were 2.89 × 104 and 4.41 × 104 M−1, while the constants of second set of binding sites were 8.56 × 104 and 1.58 × 105 M−1, respectively. The data of synchronized fluorescence spectroscopy at Δλ = 60 nm were indicative of the closer location of β-Casein-B12 complex to the Tyr residues. Additionally, the binding distance between B12 and the Trp residues of α-Casein and β-Casein were obtained in accordance to the Förster’s theory of nonradioactive energy transfer to be 1.95 nm and 1.85 nm, respectively. Relatively, the RLS results demonstrated the production of larger particles in both systems, while the outcomes of zeta potential confirmed the formation of α-Casein-B12 and β-Casein-B12 complexes and approved the existence of electrostatic interactions. We also evaluated the thermodynamic parameters by considering the fluorescence data at three varying temperatures. According to the nonlinear Stern-Volmer plots of α-Casein and β-Casein in the presence of B12 in binary systems, the two sets of binding sites indicated the detection of two types of interaction behaviors. Time-resolved fluorescence results revealed that the fluorescence quenching of complexes are static mechanism. Furthermore, the outcomes of circular dichroism (CD) represented the occurrence of conformational changes in α-Casein and β-Casein upon their binding to B12 as the binary system. The experimental results that were obtained throughout the binding of α-Casein-B12 and β-Casein-B12 complexes were confirmed by molecular modeling. Communicated by Ramaswamy H. Sarma

Tyrosine Nitroxidation Does Not Affect the Ability of α-Synuclein to Bind Anionic Micelles, but It Diminishes Its Ability to Bind and Assemble Synaptic-like Vesicles.

Parkinson's disease (PD) is characterized by dopaminergic neuron degeneration and the accumulation of neuronal inclusions known as Lewy bodies, which are formed by aggregated and post-translationally modified α-synuclein (αS). Oxidative modifications such as the formation of 3-nitrotyrosine (3-NT) or di-tyrosine are found in αS deposits, and they could be promoted by the oxidative stress typical of PD brains. Many studies have tried to elucidate the molecular mechanism correlating nitroxidation, αS aggregation, and PD. However, it is unclear how nitroxidation affects the physiological function of αS. To clarify this matter, we synthetized an αS with its Tyr residues replaced by 3-NT. Its study revealed that Tyr nitroxidation had no effect on either the affinity of αS towards anionic micelles nor the overall structure of the micelle-bound αS, which retained its α-helical folding. Nevertheless, we observed that nitroxidation of Y39 lengthened the disordered stretch bridging the two consecutive α-helices. Conversely, the affinity of αS towards synaptic-like vesicles diminished as a result of Tyr nitroxidation. Additionally, we also proved that nitroxidation precluded αS from performing its physiological function as a catalyst of the clustering and the fusion of synaptic vesicles. Our findings represent a step forward towards the completion of the puzzle that must explain the molecular mechanism behind the link between αS-nitroxidation and PD.

Modulation of the Fibrillation Kinetics and Morphology of a Therapeutic Peptide by Cucurbit[7]uril.

Fibrillation is a challenge commonly encountered in the formulation and development of therapeutic peptides. Cucurbit[7]urils (CB[7]), a group of water soluble macrocycles, have been reported to suppress fibrillation in insulin and human calcitonin through association with Phe and Tyr residues which drive fibril formation. Here, we report the effect of CB[7] on the fibrillation behavior of the HIV fusion inhibitor enfuvirtide (ENF) that contains N-terminal Tyr and C-terminal Phe residues. Thioflavin T fluorescence, CD spectroscopy, and transmission electron microscopy were used to monitor fibrillation behavior. Fibrillation onset showed a strong pH dependency, with pH 6.5 identified as the condition most suitable to monitor the effects of CB[7]. Binding of CB[7] to wild-type ENF was measured by isothermal titration calorimetry and was consistent with a single site (Ka = 2.4 × 105 M-1). A weaker interaction (Ka = 2.8 × 103 M-1) was observed for an ENF mutant with the C-terminal Phe substituted for Ala (ENFm), suggesting that Phe was the specific site for CB[7] recognition. The onset of ENF fibrillation onset was delayed, rather than fully suppressed, in the presence of CB[7]. The ENFm mutant showed a greater delay in fibrillation onset but with no observable effect on fibrillation kinetics in the presence of CB[7]. Interestingly, ENF/CB[7] and ENFm fibrils exhibited comparable morphologies, differing from those observed for ENF alone. The results indicate that CB[7] is capable of modulating fibrillation onset and the resulting ENF fibrils by specifically binding to the C-terminal Phe residue. The work reinforces the potential of CB[7] as an inhibitor of fibrillation and highlights its role in determining fibril morphologies.

Teraryl Braces in Macrocycles: Synthesis and Conformational Landscape Remodeling of Peptides.

The three-dimensional structure of medium-sized cyclic peptides accounts for their biological activity and other important physiochemical properties. Despite significant advances in the past few decades, chemists' ability to fine-tune the structure, in particular, the backbone conformation, of short peptides made of canonical amino acids is still quite limited. Nature has shown that cross-linking the aromatic side chains of linear peptide precursors via enzyme catalysis can generate cyclophane-braced products with unusual structures and diverse activities. However, the biosynthetic path to these natural products is challenging to replicate in the synthetic laboratory using practical chemical modifications of peptides. Herein, we report a broadly applicable strategy to remodel the structure of homodetic peptides by cross-linking the aromatic side chains of Trp, His, and Tyr residues with various aryl linkers. The aryl linkers can be easily installed via copper-catalyzed double heteroatom-arylation reactions of peptides with aryl diiodides. These aromatic side chains and aryl linkers can be combined to form a large variety of assemblies of heteroatom-linked multi-aryl units. The assemblies can serve as tension-bearable multijoint braces to modulate the backbone conformation of peptides as an entry to previously inaccessible conformational space.

Comprehending the intermolecular interaction of dacomitinib with bovine serum albumin: experimental and theoretical approaches

Dacomitinib (DAC), as a member of tyrosine kinase inhibitors is primarily used to treat non-small cell lung cancer. The intermolecular interaction between DAC and bovine serum albumin (BSA) was comprehended with the help of experiments and theoretical simulations. The outcomes indicated that DAC quenched the endogenous fluorescence of BSA through static quenching mode. In the binding process, DAC was preferentially inserted into the hydrophobic cavity of BSA subdomain IA (site III), and a fluorescence-free DAC-BSA complex with molar ratio of 1:1 was generated. The outcomes confirmed that DAC had a stronger affinity on BSA and the non-radiative energy transfer occurred in the combination process of two. And, it can be inferred from the outcomes of thermodynamic parameters and competition experiments with 8-aniline-1-naphthalenesulfonic acid (ANS) and D-(+)- sucrose that hydrogen bonds (H-bonds), van der Waals forces (vdW) and hydrophobic forces had a significant impact in inserting DAC into the hydrophobic cavity of BSA. The outcomes from multi-spectroscopic measurements that DAC could affect the secondary structure of BSA, that was, α-helix content decreased slightly from 51.0% to 49.7%. Moreover, the combination of DAC and BSA led to a reduction in the hydrophobicity of the microenvironment around tyrosine (Tyr) residues in BSA while had little influence on the microenvironment of around tryptophan (Trp) residues. The outcomes from molecular docking and molecular dynamics (MD) simulation further demonstrated the insertion of DAC into site III of BSA and hydrogen energy and van der Waals energy were the dominant energy of DAC-BSA stability. In addition, the influence of metal ions (Fe3+, Cu2+, Co2+, etc.) on the affinity of the system was explored. Communicated by Ramaswamy H. Sarma

Characterization of a Potent and Orally Bioavailable Lys-Covalent Inhibitor of Apoptosis Protein (IAP) Antagonist.

We have recently reported on the use of aryl-fluorosulfates in designing water- and plasma-stable agents that covalently target Lys, Tyr, or His residues in the BIR3 domain of the inhibitor of the apoptosis protein (IAP) family. Here, we report further structural, cellular, and pharmacological characterizations of this agent, including the high-resolution structure of the complex between the Lys-covalent agent and its target, the BIR3 domain of X-linked IAP (XIAP). We also compared the cellular efficacy of the agent in two-dimensional (2D) and three-dimensional (3D) cell cultures, side by side with the clinical candidate reversible IAP inhibitor LCL161. Finally, in vivo pharmacokinetic studies indicated that the agent was long-lived and orally bioavailable. Collectively our data further corroborate that aryl-fluorosulfates, when incorporated correctly in a ligand, can result in Lys-covalent agents with pharmacodynamic and pharmacokinetic properties that warrant their use in the design of pharmacological probes or even therapeutics.

New Insights on the Activity and Selectivity of MAO-B Inhibitors through In Silico Methods.

To facilitate the identification of novel MAO-B inhibitors, we elaborated a consolidated computational approach, including a pharmacophoric atom-based 3D quantitative structure-activity relationship (QSAR) model, activity cliffs, fingerprint, and molecular docking analysis on a dataset of 126 molecules. An AAHR.2 hypothesis with two hydrogen bond acceptors (A), one hydrophobic (H), and one aromatic ring (R) supplied a statistically significant 3D QSAR model reflected by the parameters: R2 = 0.900 (training set); Q2 = 0.774 and Pearson's R = 0.884 (test set), stability s = 0.736. Hydrophobic and electron-withdrawing fields portrayed the relationships between structural characteristics and inhibitory activity. The quinolin-2-one scaffold has a key role in selectivity towards MAO-B with an AUC of 0.962, as retrieved by ECFP4 analysis. Two activity cliffs showing meaningful potency variation in the MAO-B chemical space were observed. The docking study revealed interactions with crucial residues TYR:435, TYR:326, CYS:172, and GLN:206 responsible for MAO-B activity. Molecular docking is in consensus with and complementary to pharmacophoric 3D QSAR, ECFP4, and MM-GBSA analysis. The computational scenario provided here will assist chemists in quickly designing and predicting new potent and selective candidates as MAO-B inhibitors for MAO-B-driven diseases. This approach can also be used to identify MAO-B inhibitors from other libraries or screen top molecules for other targets involved in suitable diseases.

Spectroscopic, voltammetric and computational approaches shed light on the combination characteristics of an anticancer agent, bexarotene with human serum albumin

The combination process of bexarotene (BEX) with the primary transporter protein in blood plasma, human serum albumin (HSA) was explored to comprehend the anticancer agent’s transport in the circulatory system. The HSA fluorescence spectrum was discovered to be successively quenched by BEX. Involvement of static quenching process as well as the BEX–HSA complexation were suggested by the bimolecular quenching rate constant (kq) values. This finding was further corroborated by ultraviolet absorption spectral analysis. The binding affinity of BEX–HSA interaction was found as moderate on the basis of Ka values (Ka = 3.19–8.72 × 104 M−1) for the system. Thermodynamic particulars (ΔS = 175.93 J mol−1 K−1; ΔH = 25.85 kJ mol−1) revealed that the stabilizing intermolecular forces associated in the reaction were hydrophobic interactions and hydrogen bonds. BEX binding altered the microenvironment near Trp and Tyr residues of HSA, but it improved the protein’s resistance to heat stress. Both site-specific ligand displacement and computational docking outcomes indicated that Sudlow’s Site II was detected as the position for BEX binding to HSA with higher priority. The BEX–HSA complex constancy was demonstrated by the complex maintaining equilibrium during the course of the simulations, according to analysis of the molecular dynamics simulation results.

In vitro interactions of two pesticides, propazine and quinoxyfen with bovine serum albumin: Spectrofluorometric and molecular docking investigations

Binding mechanisms of two selected pesticides, propazine (PRO) and quinoxyfen (QUI) with bovine serum albumin (BSA) was examined using fluorescence, absorption and molecular docking methods. Intrinsic fluorescence of BSA was quenched in the presence of both PRO and QUI. The quenching was ascertained to be conversely linked to temperature, which suggested the contribution of static quenching process in the PRO–BSA and QUI–BSA complex formations. This results were validated by the enhancement in absorption spectrum of BSA upon binding with PRO and QUI. Binding constant values (Kf = 9.55–0.60 × 10–3 M−1 for PRO–BSA system; Kf = 7.08–5.01 × 102 M−1 for QUI–BSA system) and number of binding site (n) values for the PRO–BSA and QUI–BSA systems at different temperatures affirmed a weak binding strength with a set of equivalent binding sites on BSA. Thermodynamic data obtained for both the PRO–BSA and QUI–BSA interactions predicted that the association process was spontaneous and non-covalent contacts such as hydrophobic interactions, van der Waals forces and hydrogen bonds participated in the binding reactions. This result was further supported by the molecular docking assessments. Three-dimensional spectral results revealed the microenvironmental alterations near tryptophan (Trp) and tyrosine (Tyr) residues in BSA by the addition of PRO and QUI. The docking analysis demonstrated the binding pattern for the PRO–BSA and QUI–BSA systems and disclosed the preferred binding site of both PRO and QUI as site I (subdomain IIA) of BSA.

Control of atypical PKCι membrane dissociation by tyrosine phosphorylation within a PB1-C1 interdomain interface

Atypical PKCs are cell polarity kinases that operate at the plasma membrane where they function within multiple molecular complexes to contribute to the establishment and maintenance of polarity. In contrast to the classical and novel PKCs, atypical PKCs do not respond to diacylglycerol cues to bind the membrane compartment. Until recently it was not clear how aPKCs are recruited; whether aPKCs can directly interact with membranes or whether they are dependent on other protein interactors to do so. Two recent studies identified the pseudo-substrate region and the C1 domain as direct membrane interaction modules, however their relative importance and coupling are unknown. We combined molecular modelling and functional assays to show that the regulatory module of aPKCι, comprising the PB1 pseudo-substrate and C1 domains, forms a cooperative and spatially continuous invariant membrane interaction platform. Furthermore, we show the coordinated orientation of membrane-binding elements within the regulatory module requires a key PB1-C1 interfacial β-strand (BSL). We show this element contains a highly conserved Tyr residue that can be phosphorylated and that negatively regulates the integrity of the regulatory module, leading to membrane release. We thus expose a hitherto unknown regulatory mechanism of aPKCι membrane binding and release during cell polarization.