Conformational Changes In Protein Molecules Research Articles

Ligand-induced conformational changes in protein molecules detected by sum-frequency generation

We present the first demonstration of ligand-induced conformational changes in a biological molecule, a protein, by sum-frequency generation (SFG). Constructs of KRasG12D protein were prepared by selectively deuterating residues of a single amino acid type using isotope-labeled amino acids and cell-free protein synthesis. By attaching labeled protein to a supported bilayer membrane via a His-tag to Ni-NTA-bearing lipids, we ensured that single layers of ordered molecules were formed while preserving the protein’s native structure. Exceptionally large SFG amide I signals were produced in both labeled and unlabeled proteins, demonstrating a high degree of orientational order upon attachment to the bilayer. Deuterated protein also produced SFG signals in the CDx spectral region, which were not present in the unlabeled protein. The CDx signals were measured before and after binding a peptide inhibitor, KRpep-2d, revealing shifts in SFG intensity due to conformational changes at the labeled sites. In particular, peaks associated with CDx stretching vibrations for alanine, valine, and glycine changed substantially in amplitude upon inhibitor binding. By inspection of the crystal structure, these three residues are uniquely colocated on the protein surface in and near the nucleotide binding site, which is in allosteric communication with the site of peptide inhibitor binding, suggesting an approach to identify a ligand’s binding site. The technique offers a highly sensitive, nonperturbative method of mapping ligand-induced conformational changes and allosteric networks in biological molecules for studies of the relationship between structure and function and mechanisms of action in drug discovery.

Effect of terahertz radiation on drug activity in bacterial cells

Terahertz (THz) waves have been reported to change membrane permeability and induce conformational changes in protein molecules. Drugs action on cells involves membrane permeability, and we therefore investigated the effect of THz waves on the activity of the cytotoxic drug bleomycin on Escherichia coli. 0.46 THz radiation with an average power of 2.5 W/cm2 was noncytotoxic to E. coli cells. However, 0.46 THz radiation enhanced the cytotoxic activity of bleomycin in E. coli cells, and the drug-enhancing effect depended on the power density of the THz waves. Then, the activity of the drug remaining in the culture medium after THz radiation did not differ from that remaining after non-radiation. This indicates that THz radiation does not affect the bacterial cell-membrane permeability to bleomycin. Thus, these results suggest that 0.46 THz radiation enhances the cytotoxicity of bleomycin to E. coli cells and may influence the mechanism of bleomycin action within cells rather than affecting drug uptake.

Comparison of effects of potassium iodide and potassium chloride on structure and physicochemical properties of proteins

The effects of KI (1.25, 6.25, 12.50 mM) on the structure and physicochemical properties of proteins (bovine serum albumin, ovalbumin, and lysozyme protein) at the corresponding pH values were investigated. The results showed that the interaction of I− with protein molecules was significantly stronger than that of Cl− under different concentration conditions. The higher the concentration, the more pronounced the interaction was by turning potassium iodide into potassium iodate, which bound to proteins in a more stable form, and the conformational changes of protein molecules were more remarkable. At high concentrations, the hydrodynamic radii of the bovine serum albumin and ovalbumin proteins increased significantly, up to 20.97 % and 157.95 %, respectively, and affected the microrheological behavior of the system, weakened the tracer particle motion and the elasticity of the system, changed the microenvironment in which the particles were located, with significant changes in the secondary structure of their amide I bands. Overall, there was no significant effect on lysozyme proteins. This study offers new directions for physiological mechanisms and promotes the development and application of food products.

The time-zero HSQC method improves the linear free energy relationship of a polypeptide chain through the accurate measurement of residue-specific equilibrium constants.

EXSY (exchange spectroscopy) NMR provides the residue-specific equilibrium constants, K, and residue-specific kinetic rate constants, k, of a polypeptide chain in a two-state exchange in the slow exchange regime. A linear free energy relationship (LFER) discovered in a log k versus log K plot is considered to be a physicochemical basis for smooth folding and conformational changes of protein molecules. For accurate determination of the thermodynamic and kinetic parameters, the measurement bias arising from state-specific differences in the R1 and R2 relaxation rates of 1H and other nuclei in HSQC and EXSY experiments must be minimized. Here, we showed that the time-zero HSQC acquisition scheme (HSQC0) is effective for this purpose, in combination with a special analytical method (Π analysis) for EXSY. As an example, we applied the HSQC0 + Π method to the two-state exchange of nukacin ISK-1 in an aqueous solution. Nukacin ISK-1 is a 27-residue lantibiotic peptide containing three mono-sulfide linkages. The resultant bias-free residue-based LFER provided valuable insights into the transition state of the topological interconversion of nukacin ISK-1. We found that two amino acid residues were exceptions in the residue-based LFER relationship. We inferred that the two residues could adopt special conformations in the transition state, to allow the threading of some side chains through a ring structure formed by one of the mono-sulfide linkages. In this context, the two residues are a useful target for the manipulation of the physicochemical properties and biological activities of nukacin ISK-1.

Grass pea (Lathyrus sativus L.) protein yield and functionality as affected by extraction method: Alkaline, ultrasound‐assisted, and ultrasound pretreatment extraction

AbstractBackground and ObjectivesPlant protein products are now considered sustainable substitutes for animal proteins in new product formulations, and various methods such as ultrasound have been used to improve their properties. The aim of this study was to investigate the physicochemical and functional properties of grass pea (Lathyrus sativus L.) protein isolate was extracted, as a promising source of plant‐based protein, using conventional (CP), pre‐ultrasonic (PUP) and ultrasound‐assisted (UAP) treatments.FindingsExtraction yield, protein content, and protein yield of ultrasound treatments were increased. Ultrasound treatments improved the functional properties of pea protein. Thermal and surface properties were also altered due to the conformational changes in protein molecules during sonication. According to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, there were no differences in protein fractions of different samples in terms of molecular weight, and protein fractions between 23 and 100 kDa were observed. However, Fourier transform infrared spectroscopy results revealed changes in different treatments suggesting that sonication changed protein secondary structures. Correspondingly, optical and fluorescence microscopy images of protein solutions and foams revealed changes in the structure of proteins and CP presented larger protein aggregates and larger foam bubble sizes than PUP and UAP.ConclusionsUltrasound treatment could increase extraction and improve the functionality of proteins which was attributed to changes in the secondary structure of proteins.Significance and NoveltyGrass pea protein isolate can be considered a novel source of valuable protein and ultrasound treatment could enhance the functionality of the protein increasing potential applications of this ingredient in the food industry.

Numerical study on mechanisms of soy protein as a functional modifier for polymer materials

In the past decades, to meet the increasing demands for protecting the environments, taking bio-degradable green materials as alternatives to traditional petroleum-based polymer materials has become a global interest. From the previous studies, it is demonstrated that soy protein-based biomaterial is one of the promising candidates to be used as functional modifier for polymers, due to the complex structures of protein and diverse interactions with polymers. However, the underlying mechanisms remain unclear. Molecular dynamic (MD) simulation is a power tool and can provide insights in understanding the conformational changes of protein molecules, as well as the interactions between proteins and polymers. In this study, 11S molecule of soy protein is chosen as a representative, and three different denaturation processes are applied via MD simulation, including heat denaturation at two temperatures and the breaking of disulfide bonds. It is observed that by controlling the denaturation conditions, the structures and properties of the protein molecules can be manipulated. Low temperature condition has limited impacts on the structures and properties of 11S molecules; high temperature process, on the other hand, is found to lead to an intensive secondary phase transition of the molecules; moreover, by breaking the disulfide bonds, the hydrophobic residues of the molecules can be largely exposed, potentially forming strong interactions with the hydrophobic poly(vinylidene fluoride) (PVDF) polymer. Besides, different protein-polymer interactions could result in various property-modification effects on PVDF polymer. For instance, due to the strong interaction between PVDF and the protein molecule denatured without disulfide bonds, the dipole moment of PVDF at the interface is largely enhanced, and the polarizability under external electric fields is also improved, which is in agreement with experimental result.

Ubiquitin and regulation of apoptosis in Jurkat cells

Introduction.One of the crucial tasks in medicine is studying the molecular mechanisms of selective management of tumor cell apoptosis following conformational changes in protein molecules (ubiquitination).The purpose of the study. The aim of the project is to establish the role of ubiquitin and ubiquitinligase in dexamethasone-induced apoptosis in Jurkat cells.Materials and methods.The study was carried out on the Jurkat tumor cell line (intact cells and cells cultured in the presence of an apoptosis inducer dexamethasone in the final concentration of 10 µmol. In intact and dexamethasone-affected Jurkat cells, implementation of apoptosis and the amount of FAS-, TNF Receptor 1 and cells with reduced mitochondrial membrane potential were assessed by flow cytometry using FITC-conjugated Annexin V and Propidium Iodide. The levels of NF-κB, Apaf-1, ubiquitin and ubiquitin ligase were determined by Western blot analysis. The activity of caspase-3 was measured by spectrofluorometry.Results.When adding the apoptosis inducer dexamethasone to the Jurkat cell culture, we registered a fall in the concentration of ubiquitin and a rise in the level of ubiquitinligase against the backdrop of activated receptor(an increase in the amount of Annexin V positive cells, FASand TNF Receptor 1) and mitochondrialmediated (an increase in the number of cells with reduced mitochondrial membrane potential and elevation of Apaf-1 level) pathways of apoptosis, as opposed to the intact cell culture. We estimated the completion of apoptosis by determining the activity of caspase-3 in the investigated tumor cells.Conclusion.The obtained findings allow the conclusion that ubiquitination of regulatory and effector proteins in programmed cell death is one of the molecular mechanisms that regulates and selectively controls apoptosis in Jurkat cells.

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (km and Vmax) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

The effect of cryomechanodestruction on activation of heteropolysaccaride-protein nanocomplexes when developing nanotechnologies of plant supplements

The regularities and mechanisms of the effect of deep processing of plant raw materials were established, such as finely dispersed grinding in developing nanotechnology of obtaining frozen nanopuree and nanopowders on the transformation of bound amino acids of protein to free soluble form by меchanolysis of molecules of protein (by 45…55 % to separate α­amino acids). We discovered the mechanism of mechanodestruction of protein molecules and its nanocomplexes with other biopolymers and BAS, which is linked to mechanocracking. In the deep processing of plant raw materials, in particular, Jerusalem artichoke, which is based on the comprehensive action of cryogenic «shock» freezing, freeze drying and finely dispersed grinding processes when obtaining nanopowders, the processes of cryodestruction, mechanodestruction and mechanochemistry occur that lead to the fuller extraction of BAS from the raw material (by 1.8…2.3 times more than is in the original raw material) and destruction of biopolymers (inulin, proteins) to their monomers. It was found that the freezing and cryomechanodestruction lead to the transformation of chemical substances of Jerusalem artichoke (cryomechanochemistry) and transformation, in particular, conformational changes of protein molecules: reduction of radius of the volume of a protein molecule, radius of its nucleus, and also to a decrease in the indicator of filling the nucleaus with hydrophobic remains of amino acids. In addition, the shape of protein molecules changes. We proposed and designed cryogenic nanotechnology of finely dispersed frozen nanopuree and nanopowders from Jerusalem artichoke with prebiotic properties. It was shown that nanosupplements exceed the known world analogues in the content of BAS and dispersed composition. In addition, a large part of the substances (both BAS and biopolymers) is in the nanodimensional form.

DEVELOPMENT OF THE NEW METHOD OF THE MELTED CHEESE PRODUCTS WITHOUT SALT-MELTERS USING CRYOMECHANOLYSIS

The aim of the work is elaboration of the principally new method of deep processing of rennet cheeses to the melting using the complex action of freezing and cryomechanolysis on the raw material that gives a possibility to destruct the hardly soluble biopolymers and to transform them into soluble form. The principally new method of the deep processing of rennet cheeses for receiving the melt cheese products without salts-smelters was elaborated. It differs from the traditional ones by the complete exclusion of the salts-smelters. This method is based on the use of the influence of freezing and fine-dispersed comminution on the raw material. It allows open biological potential of the rennet cheeses more fully and to extract the hidden (bound) protein forms from nanocomplexes of lipids and mineral substances. It allows destruct the proteins of rennet cheeses to the separate polymers and dipeptides and tripeptides. The used technological methods gave a possibility to exclude the salts-smelters at the rennet cheeses manufacturing. They favor the transformation of lipid-proteins paracaseinate calcium phosphate complexes to the separate amino acids and peptides and allow receive homogenous plastic mass. It was established, that at the complex action of freezing and fine-dispersed comminution on the rennet cheese the destruction of hardly soluble lipid-protein nanocomplexes and release of protein from the bound state into free one – nanoform (by 33,5…35 % more) takes place. The mechanisms of this process, connected with cryomechanodestruction of connections between lipids and proteins and non-fermented catalysis of nanocomplexes were described. It was established, that cryomechanodestruction and non-fermented catalysis of protein to the separate monomers – α-amino acids (by 55…60 %) takes place at freezing and fine-dispersed comminution of rennet cheese before melting. The mechanism of freezing and non-fermented analysis, connected with cryomechanocracking of protein molecules at the expanse of peptide protein connections destruction to the separate α-amino acids and their transformation into the free form was described. It was also demonstrated, that the conformational changes of protein molecules take place synchronously. The offered and elaborated nanotechnology of melt cheese products on the base of rennet cheeses without salts-smelters includes complex action of freezing and fine-dispersed comminution. The mechanisms of processes, connected with cryomechanodestruction of connections between lipids and protein to the separate α-amino acids are described. The cheese fillings for “Pancake” confectionary and cheese snacks – falafels were manufactured on the base of cheese mass, received using the new method and enriching vegetable nanoadditives. They exceed the well-known analogs by chemical composition and are remarkable for the storage life, increased in 2 times. At the same time the significant part of substances (BAS and biopolymers) in cheese filings is in nanodimensional form (55…60 % of protein), especially, free α-amino acids, easily assimilated by the human organism. The recipes and technologies of sauces-dressings, sauces-deeps, cheese snacks and so on are also elaborated on the base of cheese mass, received by the new method.

Analysis of binding ability of two tetramethylpyridylporphyrins to albumin and its complex with bilirubin

An interaction between 5,10,15,20-tetrakis-(N-methyl-x-pyridyl)porphyrins, x=2; 4 (TMPyPs) with bovine serum albumin (BSA) and its bilirubin (BR) complex was investigated by UV–Viz and fluorescence spectroscopy under imitated physiological conditions involving molecular docking studies. The parameters of forming intermolecular complexes (binding constants, quenching rate constants, quenching sphere radius etc.) were determined. It was showed that the interaction between proteins and TMPyPs occurs via static quenching of protein fluorescence and has predominantly hydrophobic and electrostatic character. It was revealed that obtained complexes are relatively stable, but in the case of TMPyP4 binding with proteins occurs better than TMPyP2. Nevertheless, both TMPyPs have better binding ability with free protein compared to BRBSA at the same time. The influence of TMPyPs on the conformational changes in protein molecules was studied using synchronous fluorescence spectroscopy. It was found that there is no competition of BR with TMPyPs for binging sites on protein molecule and BR displacement does not occur. Molecular docking calculations have showed that TMPyPs can bind with albumin via tryptophan residue in the hydrophilic binding site of protein molecule but it is not one possible interaction way.

Watching Conformational Changes in Proteins by Molecular Dynamics Simulations

Proteins are dynamical molecules and their ability to adopt alternative conformations is central to their biological function. Examples include motions that underlie allosteric regulation or ligand binding, or protein dynamics in enzymes that can modulate the overall catalytic efficiency. Protein motions can often be described as an exchange between a dominant, ground state structure and one or more minor states. The structural and biophysical properties of these transiently and sparsely populated states are, however, difficult to study, and an atomic-level description of those states is challenging. In an attempt to determine how well molecular dynamics simulations can capture slow, conformational changes in protein molecules we have studied two protein systems which are known to undergo conformational exchange on the millisecond timescale, and for which structural information is available for both major and minor states. Using enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by structural information from X-ray crystallography and NMR, we show that current force fields and sampling methods allow us to sample experimentally-determined alternative conformations with surprisingly high accuracy. In particular, we find that we can reversible sample both the ground state and minor state, at that the simulations capture the structure of the minor states also. Our simulations enable us to calculate the conformational free energy between the two states, and comparison with experiments demonstrates a high accuracy. Our simulations provide insight into the structural and biophysical properties of transiently populated minor states, and help reinterpret previous experimental measurements. Further, our results demonstrate that, at least in the two cases we have studied, modern simulation methods enable us to examine these otherwise “invisible” states of proteins and describe their structural, functional and thermodynamic properties.

The Interaction of BODIPY with bovine serum albumin and its bilirubin complex

Using fluorescence and electronic spectroscopy the interaction of boron-dipyrromethene complex (BODIPY) with bovine serum albumin (BSA) and its bilirubin macromolecular complex (BR·BSA) in aqueous solution was investigated. The interaction of BODIPY is carried out by the static quenching of protein fluorescence and is predominantly hydrophobic and electrostatic in nature. The values of the binding constants were (61.2 ± 2.8) · 103 and (6.51 ± 0.3) · 103 M−1. The interaction of BODIPY with proteins leads to the observed hypso- and bathochromic shift in BODIPY absorption band. Forster resonance energy transfer theory allowed of determing the donor-ligand distance, which were 2.88 and 2.46 nm for BSA and BR·BSA, respectively. Using synchronous fluorescence spectroscopy it was possible to reveal features of BODIPY influence on conformational changes in protein molecules. It was established that BODIPY more effectively interacts with BSA compared to BR·BSA.

Influence of terahertz laser radiation on the spectral characteristics and functional properties of albumin

The exposure of albumin (transport protein of blood serum) to laser radiation with a frequency of 3.6 THz resulted in a change in the intensity of characteristic bands in UV absorption spectra and in circular dichroism spectra. These changes depend on the exposure duration and the laser radiation power and indicate conformational changes in protein molecules.

Effect of different operating parameters on the recovery of proteins from casein whey using a rotating disc membrane ultrafiltration cell

In this study, effects of different operating parameters in ultrafiltration (UF) of casein whey in rotating disk ultrafiltration module, fitted with a 30 kD followed by a 5 kD molecular weight cut-off (MWCO) membrane have been analyzed and the benefits of membrane rotation with respect to stationary membrane module are highlighted. Prior to UF, centrifugation and microfiltration (MF) were carried out with the raw casein whey with an aim to remove the major membrane foulants like colloidal matters, suspended casein particles and lipid. The effects of pH, membrane rotation and transmembrane pressure (TMP) on UF flux and rejection were studied thoroughly, giving an emphasis on the effect of operating conditions in pressure and mass transfer controlled region as well. It was observed that solution pH was having a strong effect on UF flux and rejection for treating casein whey, which was explained in terms of prevailing monomer–dimer equilibrium of β-lactoglobulin, a major constituent of casein whey, as well as due to conformational changes of protein molecules with respect to isoelectric point. It was observed that for the 30 kD membrane, after 20 min of operation, 28.5% higher flux was obtained at pH 2.8 compared to pH 5.5 at a pressure of 490 kPa for stationary membrane, whereas the same figure for rotating membrane was 49.5%. Further, 38.7% higher UF flux at 300 rpm membrane speed compared to stationary membrane in 20 min of operation at pH 2.8 and TMP 490 kPa suggests the importance of shear across the membrane in minimizing the effects of concentration polarization. In the 5 kD membrane α-lactalbumin, which was mainly in the permeate stream from the 30 kD membrane, was separated from the other low molecular weight component of whey, such as lactose.

The Studies of Density, Apparent Molar Volume, and Viscosity of Bovine Serum Albumin, Egg Albumin, and Lysozyme in Aqueous and RbI, CsI, and DTAB Aqueous Solutions at 303.15 K

Density (rho), apparent molar volume (V(phi)), and viscosity (eta) of 0.0010 to 0.0018% (w/v) of bovine serum albumin (BSA), egg albumin, and lysozyme in 0.0002, 0.0004, and 0.0008 M aqueous RbI and CsI, and (dodecyl)(trimethyl)ammonium bromide (DTAB) solutions were obtained. The experimental data were regressed against composition, and constants are used to elucidate the conformational changes in protein molecules. With salt concentration, the density of proteins is found to decrease, and the order of the effect of additives on density is observed as CsI > RbI > DTAB. The trend of apparent molar volume of proteins is found as BSA > egg-albumin > lysozyme for three additives. In general, eta values of BSA remain higher for all compositions of RbI than that of egg-albumin for CsI and DTAB. These orders of the data indicate the strength of intermolecular forces between proteins and salts, and are helpful for understanding the denaturation of proteins.

Photon Correlation Spectroscopy Investigations of Proteins

AbstractFor Abstract see ChemInform Abstract in Full Text.

Interaction of proteins with weak amphoteric charged membrane surfaces: effect of pH

Weak amphoteric charged membranes were prepared by the graft copolymerization of poly(ethylene glycol) (PEG) derivatives with pendant ionizable groups onto polyethylene (PE) porous membranes. Two types of weak amphoteric charged membranes and two types of weak single charged membranes were prepared. The pH dependence of the protein (fluorescein isothiocyanate-labeled bovine serum albumin, FITC-BSA) adsorption onto the membranes was investigated by fluorescence spectroscopy. The interfacial charge properties of the membranes and protein were also characterized at different pH values by streaming potential and electrophoretic light scattering (ELS) measurements, respectively. The adsorbed amount onto each ionic PEG chain grafted membrane showed a uniform maximum value near the isoelectric point (IEP) of the protein (pH 4.1). On both sides of the IEP (pHs 3.3 and 7.2), the adsorption experiments and zeta ( ζ) potential measurements were well correlated: the contribution of electrostatic interaction was dominant for the protein adsorption behavior. In the alkaline condition (pH 10.2), the adsorption experiments contradict the ζ potential measurements. It suggested that the conformational change of protein molecule influenced the adsorption behavior. Finally, these results indicated the potential of controlling the protein–ionic PEG chain interaction on the membrane surfaces by the pH adjustment of the outer solution.

Conformational changes in protein molecules upon adsorption on ultrafine particles

Conformational changes in hemoglobin (a “soft” protein) and catalase (a relatively “soft” protein) upon adsorption on ultrafine silica particles (average diameter 15 nm) have been studied by circular dichroism (CD). Because the light scattering intensity of the ultrafine silica particles is very low, the secondary structure of protein molecules on solid surfaces can be estimated from the CD spectra of the suspension of the ultrafine silica particles on which proteins are adsorbed. The extent of the changes in the secondary structure of both the protein molecules was found to increase with decreasing pH and adsorbed amount. Hemoglobin showed the changes in the secondary structure up to high pH and high adsorbed amount. Therefore, soft proteins are susceptible to conformational changes upon adsorption on solid surfaces. These CD spectrum data give us some quantitative information about the conformational changes in protein molecules upon adsorption on solid surfaces.

TYROSYL FLUORESCENCE SPECTRA OF PROTEINS LACKING TRYPTOPHAN: EFFECTS OF INTRAMOLECULAR INTERACTIONS

Abstract— The fluorescence spectra of class A proteins which lack tryptophanyl residues were examined in detail, and revealed some differences in the position of maximum and half‐width of the tyrosine emission band between proteins and tyrosine itself. These parameters were affected by conformational changes in protein molecules (denaturation, thermal effects, ion binding) which also induced variations in absorption spectra and fluorescence quantum yields. These observations are thought to be related to the formation of hydrogen bonds between the hydroxyl group of the tyrosyl residues and proton acceptor groups in the protein.