Protein Conformational Changes Research Articles

Computing committors via Mahalanobis diffusion maps with enhanced sampling data.

The study of phenomena such as protein folding and conformational changes in molecules is a central theme in chemical physics. Molecular dynamics (MD) simulation is the primary tool for the study of transition processes in biomolecules, but it is hampered by a huge timescale gap between the processes of interest and atomic vibrations that dictate the time step size. Therefore, it is imperative to combine MD simulations with other techniques in order to quantify the transition processes taking place on large timescales. In this work, the diffusion map with Mahalanobis kernel, a meshless approach for approximating the Backward Kolmogorov Operator (BKO) in collective variables, is upgraded to incorporate standard enhanced sampling techniques, such as metadynamics. The resulting algorithm, which we call the target measure Mahalanobis diffusion map (tm-mmap), is suitable for a moderate number of collective variables in which one can approximate the diffusion tensor and free energy. Imposing appropriate boundary conditions allows use of the approximated BKO to solve for the committor function and utilization of transition path theory to find the reactive current delineating the transition channels and the transition rate. The proposed algorithm, tm-mmap, is tested on the two-dimensional Moro-Cardin two-well system with position-dependent diffusion coefficient and on alanine dipeptide in two collective variables where the committor, the reactive current, and the transition rate are compared to those computed by the finite element method (FEM). Finally, tm-mmap is applied to alanine dipeptide in four collective variables where the use of finite elements is infeasible.

Molecular characterization of capsaicin binding interactions with ovalbumin and casein

Capsaicin (CAP) rich diets may help with a variety of human pathophysiological conditions; however, CAP administration is difficult due to its high pungency and limited water solubility. This study comprehensively explored the mechanism of CAP binding with ovalbumin (OVA) and casein (CAS) by multi-spectroscopic, thermodynamics, and molecular docking simulation at pH 7.4, as well as the prospect of employing these food proteins as CAP carriers. The findings demonstrated that CAP could interact with OVA/CAS and increase their fluorescence intensity, which was followed by specific protein conformational changes. According to the ITC data, the interaction between CAP and OVA/CAS proceeded spontaneously, with hydrogen bonding and hydrophobic interactions governing the binding process. The binding constant Ka for the CAP-OVA and CAP-CAS complexes was determined by ITC to be 1.07 ± 0.21 ⅹ 105 M−1 and 2.22 ± 0.14 ⅹ 105 M−1, respectively. Molecular docking analysis further indicated the existence of a high affinity CAP binding site on OVA and CAS, supporting the experimental findings. Moreover, the data demonstrated that CAP binding interactions with these proteins led to the formation of complexes with about 97% CAP encapsulation efficiency, contributing to a synergistic enhancement in their antioxidant activity. Therefore, this study implies that OVA and CAS have great potential for being utilized as edible delivery vehicles for lipophilic bioactive molecules such as CAP.

Oxidized cellulose nanofibrils-based surimi gel enhancing additives: Interactions, performance and mechanisms

Surimi-based products with preferred textural properties and high nutritional value are fashionable throughout the market. Enhancing surimi gel characteristics is of key importance for enabling the following processing and warranting the end-product quality, but challenging due to the lack of energy- and performance-efficient processes and the elusive mechanisms underpinned such enhancements. Focusing on enhancing surimi gel properties by introducing sustainable additives, we successfully explore functionalized cellulose nanofibrils as a textural enhancer in surimi gels and establish a new model to expound the interaction mechanisms. Firstly, TEMPO-mediated oxidation prior to homogenization harvests functionalized cellulose nanofibrils with unique fibrillary shape, uniform nano-size, high surface hydrophilicity and activated -COO - groups, highly suited for the additive use in surimi gels (Monto et al., 2021). Secondly, the innovative application of oxidized cellulose nanofibrils at a remarkably low concentrations (0.1 g/100 g surimi) greatly improve the whiteness, microstructure, water holding capacity, gel strength and thermal stability of the surimi gels, through a synergistic effect of matrix-reinforcing, water binding and entrapping, covalent interactions (disulfide bonds, amide bonds), non-covalent interactions (hydrogen bonds, ionic bonds) and favorable protein conformational changes within the network. Next, a new mechanism model toward the high-performance surimi gels is concluded to better elucidate the interactions between oxidized cellulose nanofibrils and myosin molecules. We expect that this work can provide guidelines on designing novel and high-quality cellulose-additive foods and understanding the roles of food additives in meat gel systems, for advancing the applications of new additives and gelation mechanisms in the food industry. • A low cellulose nanofibrils (TCN) addition (0.1%) greatly improves surimi gel properties. • Deliberately activated COO − groups at TCN surface facilitates cellulose-myosin interactions. • Improved water holding capacity due to surface hydrophicility, water binding and entrapping. • Enhanced gel strength due to newly introduced ionic bonds, hydrogen bonds and physical entanglement. • A new mechanism model illustrating cellulose-protein interactions is established.

Novel trehalose-based excipients for stabilizing nebulized anti-SARS-CoV-2 antibody

COVID-19 is caused by the infection of the lungs by SARS-CoV-2. Monoclonal antibodies, such as sotrovimab, showed great efficiency in neutralizing the virus before its internalization by lung epithelial cells. However, parenteral routes are still the preferred route of administration, even for local infections, which requires injection of high doses of antibody to reach efficacious concentrations in the lungs. Lung administration of antibodies would be more relevant requiring lower doses, thus reducing the costs and the side effects. But aerosolization of therapeutic proteins is very challenging, as the different processes available are harsh and trigger protein aggregation and conformational changes. This decreases the efficiency of the treatment, and can increase its immunogenicity.To address those issues, we developed a series of new excipients composed of a trehalose core, a succinyl side chain and a hydrophobic carbon chain (from 8 to 16 carbons). Succinylation increased the solubility of the excipients, allowing their use at relevant concentrations for protein stabilization. In particular, the excipient with 16 carbons (C16TreSuc) used at 5.6 mM was able to preserve colloidal stability and antigen-binding ability of sotrovimab during the nebulization process. It could also be used as a cryoprotectant, allowing storage of sotrovimab in a lyophilized form during weeks. Finally, we demonstrated that C16TreSuc could be used as an excipient to stabilize antibodies for the treatment against COVID-19, by in vitro and in vivo assays. The presence of C16TreSuc during nebulization preserved the neutralization capacity of sotrovimab against SARS-CoV-2 in vitro; an increase of its efficacy was even observed, compared to the non-nebulized control. The in vivo study also showed the wide distribution of sotrovimab in mice lungs, after nebulization with 5.6 mM of excipient.This work brings a solution to stabilize therapeutic proteins during storage and nebulization, making pulmonary immunotherapy possible in the treatment of COVID-19 and other lung diseases.

The recovery effect of Vitamin C on structural alterations due to Streptozotocin-Induced diabetes in rat testicular tissues

Type I Diabetes is a multisystem disease that causes alterations in carbohydrate, protein, and fat metabolisms due to hyperglycemia. It has an extensive pathology, especially the mechanism involving oxidative stress is still complex. Type I diabetes is correlated with increased formation of free radicals and decreased levels of antioxidant potential. Vitamin C (Vit C) is a powerful antioxidant that participates in antioxidant defense, protecting lipid membranes and proteins from oxidative damage by donating electrons to free radicals.The effect of type I diabetes and the recovery role of Vit C on the structure and composition of the biomolecular content of testicular tissue is still unknown. Therefore, the current study aimed to investigate the alterations in the biomolecules of rat testes due to Streptozotocin (STZ)-induced type I diabetes using Attenuated Total Reflectance (ATR)-Fourier Transform Infrared (FTIR) spectroscopy and histological staining. The results revealed that the biomolecular structure and composition of testicular tissue are highly affected due to the development of diabetes. We obtained decreased saturation levels and increased unsaturation index in the lipids indicating the presence of lipid peroxidation in the diabetic state. The elevated lipid peroxidation levels have been implicated in the pathogenesis of naturally occurring and chemically induced diabetes. On the other hand, the protein content of diabetic rat testicular tissue was shown to decrease considerably, indicating an increase in proteolysis processes. Supporting the ratio of protein structural and conformational change, protein secondary structural components were also found to alter substantially in the diabetic state. Diabetes was also shown to lead to a decrease in the content of nucleic acids compared to proteins. These diabetes-induced alterations were found to be substantially recovered with the administration of Vit C. Although different doses and administration types of Vit C have been reported in the literature, there is no consensus yet. Therefore, we used three different doses of Vit C in our study as high (100 mg/kg/day), medium (50 mg/kg/day) and low (15 mg/kg/day) doses intraperitoneally in the present study, and the medium dose was found to be the most effective in the recovery from the diabetes-induced structural damages on rat testicular tissue. Vit C may have a therapeutic effect to be used as a complementary therapy in the treatment of diabetes.

CSAlign and CSAlign-Dock: Structure alignment of ligands considering full flexibility and application to protein–ligand docking

Structure prediction of protein–ligand complexes, called protein–ligand docking, is a critical computational technique that can be used to understand the underlying principle behind the protein functions at the atomic level and to design new molecules regulating the functions. Protein-ligand docking methods have been employed in structure-based drug discovery for hit discovery and lead optimization. One of the important technical challenges in protein–ligand docking is to account for protein conformational changes induced by ligand binding. A small change such as a single side-chain rotation upon ligand binding can hinder accurate docking. Here we report an increase in docking performance achieved by structure alignment to known complex structures. First, a fully flexible compound-to-compound alignment method CSAlign is developed by global optimization of a shape score. Next, the alignment method is combined with a docking algorithm to dock a new ligand to a target protein when a reference protein–ligand complex structure is available. This alignment-based docking method, called CSAlign-Dock, showed superior performance to ab initio docking methods in cross-docking benchmark tests. Both CSAlign and CSAlign-Dock are freely available as a web server at https://galaxy.seoklab.org/csalign.

Adenylate Kinase-Catalyzed Reactions of AMP in Pieces: Specificity for Catalysis at the Nucleoside Activator and Dianion Catalytic Sites.

The pressure to optimize the enzymatic rate acceleration for adenylate kinase (AK)-catalyzed phosphoryl transfer has led to the evolution of an induced-fit mechanism, where the binding energy from interactions between the protein and substrate adenosyl group is utilized to drive a protein conformational change that activates the enzyme for catalysis. The adenine group of adenosine contributes 11.8 kcal mol-1 to the total ≥14.7 kcal mol-1 adenosine stabilization of the transition state for AK-catalyzed phosphoryl transfer to AMP. The relative third-order rate constants for activation of adenylate kinase, by the C-5 truncated adenosine 1-(β-d-erythrofuranosyl)adenine (EA), for catalysis of phosphoryl transfer from ATP to phosphite dianion (HP, kcat/KHPKAct = 260 M-2 s-1), fluorophosphate (47 M-2 s-1), and phosphate (9.6 M-2 s-1), show that substitution of -F for -H and of -OH for -H at HP results, respectively, in decreases in the reactivity of AK for catalysis of phosphoryl transfer due to polar and steric effects of the -F and -OH substituents. The addition of a 5'-CH2OH to the EA activator results in a 3.0 kcal mol-1 destabilization of the transition state for AK-activated phosphoryl transfer to HP due to a steric effect. This is smaller than the 8.3 kcal mol-1 steric effect of the 5'-CH2OH substituent at OMP on HP-activated OMPDC-catalyzed decarboxylation of 1-(β-d-erythrofuranosyl)orotate. The 2'-OH ribosyl substituent shows significant interactions with the transition states for AK-catalyzed phosphoryl transfer from ATP to AMP and for adenosine-activated AK-catalyzed phosphoryl transfer from ATP to HP.

Exploring the binding mechanism of pumpkin seed protein and apigenin: Spectroscopic analysis, molecular docking and molecular dynamics simulation

Apigenin is a vital flavonoid that has attracted considerable attention in recent years due to its potent health benefits. Pumpkin seed protein (PSP) is a valuable by-product after pumpkin seed processing. However, the non-covalent interaction mechanism between apigenin and PSP remains to be explored. Here, the structural and conformational changes and molecular mechanisms underlying their interaction were investigated using multi-spectroscopic analysis integrated with molecular simulation approaches. The experimental results indicated that apigenin statically quenched the intrinsic fluorescence of PSP, indicating that the interaction between them did occur. Thermodynamic analysis and computational simulation results showed that the driving forces between apigenin and PSP were the hydrophobic interactions that predominated. The binding of apigenin resulted in changes in the conformation, microenvironment, and surface hydrophobicity of PSP. Furthermore, it was revealed by molecular docking and molecular dynamic (MD) simulations that apigenin maintained binding stability in the hydrophobic pockets of the protein through its interaction with several key residues. Additionally, it was found that the movement strength and directions of individual resides were altered after apigenin binding, which is the molecular basis of protein structural and conformational changes induced by apigenin. This study may provide theoretical evidence for the potential application of PSP and apigenin complex products in the food industry.

Engineering a Conformationally Switchable Artificial Metalloprotein.

Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3-, H3C-, and iPr-). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.

In silico and docking studies on the binding activities of Keap1 of antioxidant compounds in non-oilseed legumes

We used in silico methods to predict the physiochemical and pharmacological characteristics, toxicity, and biological activities of the screened compounds. All compounds showed positive results while calculating their physiochemical and pharmacokinetic descriptors. Using the Prediction of Activity Spectra for Substances (PASS) software on compounds form non-oilseed legumes, we identified compounds (mainly 4 polyphenol compounds) with anti-infective, anti-eczematic, antimutagenic, muco-membranous protector, fibrinolytic, anticarcinogenic, hepato-protectant, cardio-protectant, antioxidant, and astringent effect. PASS predicted HO-1 expression enhancing and free radical scavenging properties for gallic acid, coumaric acid, catechin, and epicatechin. Data about validation protocols for molecular docking of ligand IVV to Keap1 was performed by root mean square deviation (RMSD) value is used to validate docking protocol and representation mainly for analyzing stability of protein and predicting conformational changes of protein. Molecular docking is a powerful technique for studies of receptor-ligand interaction and has led to the discovery of Keap1-Nrf2 small molecule inhibitors. Keap1 inhibits the degradation of Nrf2. Our results suggest that screened compounds from non-oilseed legumes can effectively interact with the Keap1 binding site and dissociate Keap1 and Nrf2. The screened compounds from non-oilseed legumes that displayed high binding affinities with Keap1 are promising new Nrf2 activators. We performed molecular docking to identify the molecular interactions of gallic acid, catechin, and epicatechin with Keap1. Non-oilseed legumes plant is a natural source of potent antioxidant that may prevent diseases and could be potentially used as functional food, nutraceuticals, and drugs.

Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice.

Unusually low temperatures caused by global climate change adversely affect rice production. Sensing cold to trigger signal network is a key base for improvement of chilling tolerance trait. Here, we report that Oryza sativa Calreticulin 3 (OsCRT3) localized at the endoplasmic reticulum (ER) exhibits conformational changes under cold stress, thereby enhancing its interaction with CBL-interacting protein kinase 7 (OsCIPK7) to sense cold. Phenotypic analyses of OsCRT3 knock-out mutants and transgenic overexpression lines demonstrate that OsCRT3 is a positive regulator in chilling tolerance. OsCRT3 localizes at the ER and mediates increases in cytosolic calcium levels under cold stress. Notably, cold stress triggers secondary structural changes of OsCRT3 and enhances its binding affinity with OsCIPK7, which finally boosts its kinase activity. Moreover, Calcineurin B-like protein 7 (OsCBL7) and OsCBL8 interact with OsCIPK7 specifically on the plasma membrane. Taken together, our results thus identify a cold-sensing mechanism that simultaneously conveys cold-induced protein conformational change, enhances kinase activity, and Ca2+ signal generation to facilitate chilling tolerance in rice.

Structural dynamics of the N-terminal SH2 domain of PI3K in its free and CD28-bound states.

Protein conformational changes with fluctuations are fundamental aspects of protein-protein interactions (PPIs); understanding these motions is required for the rational design of PPI-regulating compounds. Src homology 2 (SH2) domains are commonly found in adapter proteins involved in signal transduction and specifically bind to consensus motifs of proteins containing phosphorylated tyrosine (pY). Here, we analysed the interaction between the N-terminal SH2 domain (nSH2) of the regulatory subunit in phosphoinositide 3-kinase (PI3K) and the cytoplasmic region of the T-cell co-receptor, CD28, using NMR and molecular dynamics (MD) simulations. First, we assigned the backbone signals of nSH2 on 1 H-15 N heteronuclear single quantum coherence spectra in the absence or presence of the CD28 phosphopeptide, SDpYMNMTPRRPG. Chemical shift perturbation experiments revealed allosteric changes at the BC loop and the C-terminal region of nSH2 upon CD28 binding. NMR relaxation experiments showed a conformational exchange associated with CD28 binding in these regions. The conformational stabilisation of the C-terminal region correlated with the regulation of PI3K catalytic function. Further, using 19 F- and 31 P-labelled CD28 phosphopeptide, we analysed the structural dynamics of CD28 and demonstrated that the aromatic ring of the pY residue fluctuated between multiple conformations upon nSH2 binding. Our MD simulations largely explained the NMR results and the structural dynamics of nSH2 and CD28 in both bound and unbound states. Notably, in addition to its major conformation, we detected a minor conformation of nSH2 in the CD28 bound state that may explain the allosteric conformational change in the BC loop.

The Effects of Ethanol and Rutin on the Structure and Gel Properties of Whey Protein Isolate and Related Mechanisms

The effects of different levels of rutin (0, 0.05%, 0.1%, 0.2% and 0.3% w/v) and ethanol on the structure and gel properties of whey protein isolate (WPI) were examined. The results showed that the addition of ethanol promoted the gel formation of WPI. The addition of rutin increased the gel strength of WPI and maintained the water-holding capacity of the gel. Ethanol caused an increase in thiol content and surface hydrophobicity, but rutin decreased the thiol content and surface hydrophobicity of WPI. The particle size, viscosity and viscoelasticity of WPI increased at rutin levels of 0.2% and 0.3%, indicating that rutin caused cross-linking and aggregation of WPI, but rutin had no significant effect on the zeta-potential, indicating that electrostatic interactions were not the main force causing the changes in protein conformation and gel properties. Ethanol and rutin improved the gel properties of WPI possibly by inducing cross-linking of WPIs via hydrophobic and covalent interactions.

Anxiolytic effect of YangshenDingzhi granules: Integrated network pharmacology and hippocampal metabolomics.

Anxiety disorder is one of the most common mental diseases. It is mainly characterized by a sudden, recurring but indescribable panic, fear, tension and/or anxiety. Yangshendingzhi granules (YSDZ) are widely used in the treatment of anxiety disorders, but its active ingredients and underlying mechanisms are not yet clear. This study integrates network pharmacology and metabolomics to investigate the potential mechanism of action of YSDZ in a rat model of anxiety. First, potential active ingredients and targets were screened by network pharmacology. Then, predictions were verified by molecular docking, molecular dynamics and western blotting. Metabolomics was used to identify differential metabolites and metabolic pathways. All results were integrated for a comprehensive analysis. Network pharmacology analysis found that Carotene, β-sitosterol, quercetin, Stigmasterol, and kaempferol in YSDZ exert anxiolytic effects mainly by acting on IL1β, GABRA1, PTGS1, ESR1, and TNF targets. Molecular docking results showed that all the affinities were lower than -5kcal/mol, and the average affinities were -7.7764kcal/mol. Molecular dynamics simulation results showed that RMSD was lower than 2.5 A, and the overall conformational changes of proteins were small, indicating that the small molecules formed stable complexes with proteins. The results of animal experiments showed that YSDZ exerts anxiolytic effects by regulating GABRA1 and TNF-α, ameliorating pathological damage in hippocampal CA1, and regulating metabolic pathways such as thiamine, cysteine and methionine metabolism, lysine biosynthesis and degradation. Altogether, we reveal multiple mechanisms through which YSDZ exerts its anti-anxiety effects, which may provide a reference for its clinical application and drug development.

Long-range allostery mediates the regulation of plasminogen activator inhibitor-1 by cell adhesion factor vitronectin

The serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable and active conformation, with a solvent-accessible reactive center loop (RCL), to a stable, inactive, or latent conformation, with the RCL inserted into the central β-sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin, which hinders the latency transition rate approximately twofold. The molecular mechanisms leading to this rate change are unclear. Here, we investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central β-sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition; this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40Å away from the binding site, thus blocking transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that were previously identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin-bound state, corresponding to a cryptic drug-binding site identified by crystallography. Overall, these results shed new light on PAI-1 latency transition regulation by vitronectin and illustrate the potential of path sampling simulations for understanding functional protein conformational changes and for facilitating drug discovery.

Binary-FRET reveals transient excited-state structure associated with activity-dependent CaMKII - NR2B binding and adaptation

Synaptic functions are mediated and modulated by a coordinated choreography of protein conformational changes and interactions in response to intracellular calcium dynamics. Time-lapse Förster resonance energy transfer can be used to study the dynamics of both conformational changes and protein-protein interactions simultaneously under physiological conditions if two resonance energy transfer reactions can be multiplexed. Binary-FRET is a technique developed to independently monitor the dynamics of calcium-calmodulin dependent protein kinase-II catalytic-domain pair separation in the holoenzyme, and its role in establishing activity-dependent holoenzyme affinity for the NR2B binding fragment of the N-methyl-D-aspartate receptor. Here we show that a transient excited-state intermediate exists where paired catalytic-domains in the holoenzyme first separate prior to subsequent NR2B association. Additionally, at non-saturating free calcium concentrations, our multiplexed approach reveals that the holoenzyme exhibits a biochemical form of plasticity, calcium dependent adaptation of T-site ligand binding affinity.

Molecular insights into the role of amylose/amylopectin ratio on gluten protein organization

Waxy (WX) and high-amylose (HA) wheat flours have interesting functional and/or nutritional characteristics, but low technological properties compared to regular wheat. Here a set of three wheat lines, having different amylose content but sharing the same varietal background, were compared to shed light on the role of the amylose/amylopectin ratio on the protein conformational changes that lead to gluten formation. Despite the absence of differences in their protein profile, as also confirmed by thiolomic approaches, both WX and HA lines developed a weaker gluten than the control sample. The altered amylose/amylopectin ratio exerts a matrix effect establishing a competition for water with proteins, leading to a different protein structure and three-dimensional organization of the gluten network. These results add a piece to the understanding of the molecular aspects that oversee matrix effects on gluten formation in wheat, which description can be helpful for a rational optimization of the transformation process.

The investigation of the effects of postnatal alcohol exposure on molecular content and antioxidant capacity of mice liver tissue

One of the most common causes of fetal alcohol spectrum disease (FASD) characterized with neurodevelopmental disorder and growth retardation, is the postnatal alcohol consumption. Since studies in literature are mainly focused on alcohol-induced effects on brain tissues, the molecular effects of postnatal alcohol consumption on fetal liver are not clarified yet. The aim of this study is to determine the postnatal alcohol consumption-induced structural and compositional changes on liver tissue and the antioxidant capacity of liver. Newborn mice were divided into 3 groups as control group without any treatment, alcohol group treated with 3.0 g/kg of ethanol in 0.02 ml/g of artificially enriched milk between Postnatal Days (PD) 3–20 and intragastric intubation control group which was intragastrically intubated in the same method as the alcohol group but without ethanol/milk. These postnatal days in mice refers prenatal period (third trimester) of gestation in human. The biomolecular changes were determined by ATR-FTIR spectral analysis of the samples, besides the biochemical measurement of total protein content and antioxidant capacity of liver tissue. The result of the current study shows that while there was a slight increase in total lipid content, significant decrease in unsaturated lipid and total protein contents and total antioxidant capacity of liver were observed in alcohol-treated group. Thus, it is concluded that postnatal alcohol treatment causes significant changes in tissue proteins and lipids by inducing lipid peroxidation and changes in protein conformations of the liver tissue. In addition to that alcohol consumption also reduce the antioxidant capacity of liver tissue.

Scanning pH-metry for Observing Reversibility in Protein Folding.

One of the main factors affecting protein structure in solution is pH. Traditionally, to study pH-dependent conformational changes in proteins, the concentration of the H+ ions is adjusted manually, complicating real-time analyses, hampering dynamic pH regulation, and consequently leading to a limited number of tested pH levels. Here, we present a programmable device, a scanning pH-meter, that can automatically generate different types of pH ramps and waveforms in a solution. A feedback loop algorithm calculates the required flow rates of the acid/base titrants, allowing one, for example, to generate periodic pH sine waveforms to study the reversibility of protein folding by fluorescence spectroscopy. Interestingly, for some proteins, the fluorescence intensity profiles recorded in such a periodically oscillating pH environment display hysteretic behavior indicating an asymmetry in the sequence of the protein unfolding/refolding events, which can most likely be attributed to their distinct kinetics. Another useful application of the scanning pH-meter concerns coupling it with an electrospray ionization mass spectrometer to observe pH-induced structural changes in proteins as revealed by their varying charge-state distributions. We anticipate a broad range of applications of the scanning pH-meter developed here, including protein folding studies, determination of the optimum pH for achieving maximum fluorescence intensity, and characterization of fluorescent dyes and other synthetic materials.

Electrodetection of Small Molecules by Conformation-Mediated Signal Enhancement.

Electrochemical biosensors allow the rapid, selective, and sensitive transduction of critical biological parameters into measurable signals. However, current electrochemical biosensors often fail to selectively and sensitively detect small molecules because of their small size and low molecular complexity. We have developed an electrochemical biosensing platform that harnesses the analyte-dependent conformational change of highly selective solute-binding proteins to amplify the redox signal generated by analyte binding. Using this platform, we constructed and characterized two biosensors that can sense leucine and glycine, respectively. We show that these biosensors can selectively and sensitively detect their targets over a wide range of concentrations-up to 7 orders of magnitude-and that the selectivity of these sensors can be readily altered by switching the bioreceptor's binding domain. Our work represents a new paradigm for the design of a family of modular electrochemical biosensors, where access to electrode surfaces can be controlled by protein conformational changes.