Protein denaturation in vacuo: intrinsic unfolding pathways associated with the native tertiary structure of lysozyme

Using computer-simulated molecular dynamics, we study the effect of sequence mutation on the unfolding mechanism of a native fold. The system considered is the native fold of hen egg-white lysozyme, exposed to centrifugal unfolding in vacuo. This unfolding bias elicits configurational transitions that imitate the behaviour of anhydrous proteins diffusing after electrospraying from neutral-pH solutions. By changing the sequences threaded onto the native fold of lysozyme, we probe the role of disulfide bridges and the effect of a global mutation. We find that the initial denaturing steps share common characteristics for the tested sequences. Recurrent features are: (i) the presence of dumbbell conformers with significant residual secondary structure, (ii) the ubiquitous formation of hairpins and two-stranded β-sheets regardless of disulfide bridges, and (iii) an unfolding pattern where the reduction in folding complexity is highly correlated with the decrease in chain compactness. These findings appear to be intrinsic to the shape of the native fold, suggesting that similar unfolding pathways may be accessible to many protein sequences.     

Curcumin as a tool to assess the surface hydrophobicity of proteins

ABSTRACT

Curcumin is non-fluorescent in aqueous media; however, in the vicinity of hydrophobic surface it fluoresces. This property is used to assess the use of curcumin as a surface hydrophobic probe. The surface hydrophobicity of proteins was measured by calculating the binding affinity and surface hydrophobicity index value. Surface hydrophobicity of bovine serum albumin, β-lactoglobulin, soy lipoxygenase-1, ovalbumin, and lysozyme is in the following order: bovine serum albumin?>?β-lactoglobulin?>?soy lipoxygenase-1?>?ovalbumin?>?lysozyme. The binding affinities of curcumin decreased with the decrease in surface hydrophobicity of proteins. The orders of surface hydrophobicity index value, determined using curcumin, show similar trend with the reported values of standard probes, viz. cis-parinaric acid and 1-anilinonaphthalene-8-sulfonate. The surface hydrophobicity index value of proteins determined using curcumin decreased in the presence of urea, suggesting the possible use of curcumin as a probe to determine the surface hydrophobicity of proteins.     

An Equilibrium and a Kinetic Stopped-Flow Fluorescence Study of the Binding of Various Metal Ions to Goat Alpha-Lactalbumin

Various metal ions bind to the protein α-lactalbumin prepared from goat milk. The stability of the protein after metal binding is compared with that of the apo-protein by monitoring the fluorescence of the tryptophan residues under equilibrium conditions. The kinetics of the metal binding is studied by stopped-flow fluorescence spectroscopy. By means of the Arrhenius plots, the activation energy with regard to the binding of the different ions is determined.     

Interaction of hen egg white lysozyme with cefpiramide sodium: multi-spectroscopic and computational simulations

Abstract

Under simulated physiological conditions (pH?=?7.40), the interaction between cefpiramide sodium and hen egg white lysozyme was studied with multi-spectroscopy and molecular docking. The results showed that cefpiramide sodium quenched the fluorescence of hen egg white lysozyme by static quenching, and the number of binding site n was about 1. The binding distance (r) between cefpiramide sodium and hen egg white lysozyme was obtained based on the Förster nonradioactive resonance energy transfer and r was less than 7?nm, which indicated that there was a non-radiative energy transition in the system. The thermodynamic parameters were obtained from the van't Hoff equation, and the Gibbs free energy ΔG?H?S?>?0, indicating hydrophobic interaction played a major role in forming the cefpiramide sodium-hen egg white lysozyme complex. Synchronous spectra, circular dichroism spectra and UV-Vis spectra showed that cefpiramide sodium changed the conformation of hen egg white lysozyme. The molecular docking results showed that the binding position of cefpiramide sodium was close to the active center composed of Asp52 and Glu35 residues, suggesting that cefpiramide sodium could change the microenvironment of amino acid residues at the catalytic active center of hen egg white lysozyme.     

Maternal obesity does not influence human milk protein 15N natural isotope abundance

ABSTRACT

Obesity increases protein metabolism with a potential effect on nitrogen isotope fractionation. The aim of this study was to test the influence of obesity on human milk extracted protein ¹⁵N natural isotope abundance (NIA) at one month post-partum and to compare human milk extracted protein ¹⁵N NIA and bulk infant hair ¹⁵N NIA. This cross-sectional observational study involved 16 obese mothers (body mass index (BMI)?≥?30?kg?m^?2 before pregnancy) matched with 16 normal-weight mothers (18.5?kg?m^?2?≤?BMI?<?25?kg?m^?2) for age and pregnancy characteristics. Human milk extracted protein and bulk infant hair ¹⁵N NIA were determined by isotope ratio monitoring by mass spectrometry interfaced to an elemental analyser (IRM-EA/MS). No significant difference was found in human milk protein ¹⁵N NIA values between obese and normal-weight mothers (8.93?±?0.48?‰ vs. 8.95?±?0.27?‰). However, human milk protein ¹⁵N NIA was significantly lower than bulk infant hair ¹⁵N NIA: 8.94?±?0.38?‰ vs. 9.66?±?0.69?‰, respectively. On the basis of these results, it is concluded that human milk protein ¹⁵N NIA measured at one month post-partum is not influenced by maternal obesity. These findings suggest that ¹⁵N NIA may be exploited to study metabolism without considering maternal obesity as a confounder.     

Interaction of chlorogenic acid with milk proteins analyzed by spectroscopic and modeling methods

Polyphenols are powerful antioxidants implicated in reducing the risk of human cancer and cardiovascular disease. Chlorogenic acid is a principal polyphenol in coffee, a beverage consumed worldwide on an immense scale. In many countries, coffee is consumed with milk, which has been shown to affect the bioavailability of polyphenols. Here we assessed the interactions of chlorogenic acid with five major milk proteins (α-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin) by spectroscopy and molecular docking calculations. The data showed that the number of binding sites in each chlorogenic acid–milk protein complex was close to 1. Fluorescence quenching of milk proteins by chlorogenic acid occurred through a static mechanism and the binding distance was smaller than 8 nm. Binding constant for chlorogenic acid–β-lactoglobulin was larger than those for chlorogenic acid-α-lactalbumin, chlorogenic acid–α-casein, chlorogenic acid–β-casein, and chlorogenic acid–κ-casein. Thermodynamic parameters revealed that Van der Waals forces and hydrogen bond interactions predominated in chlorogenic acid–α-casein and chlorogenic acid–α-lactalbumin complexes; hydrophobic interactions were predominant in chlorogenic acid–β-casein, chlorogenic acid–κ-casein, and chlorogenic acid–β-lactoglobulin. Molecular docking calculations identified chlorogenic acid was located near Pse66, Ile65, and Pro29 in the chlorogenic acid–α-casein adduct, Leu138, Thr126, Gln123, Ser124, Gln167, Ser166, Ser168 in the chlorogenic acid–β-casein adduct, Glu147, Asn123, Val143, Pse149, Pro120, Leu79, Ala148 in the chlorogenic acid–κ-casein adduct, Glu49, Tyr103, Gln54, His32, Trp104, Leu110 in the chlorogenic acid–α-lactalbumin adduct, and Cys66, Leu22, Lys60, Trp61, Ser21, Lys69, Gln59 in the chlorogenic acid–β-lactoglobulin adduct. Notably, the antioxidant activity of chlorogenic acid decreased significantly on interaction with each of the milk proteins (p < 0.05). This study reveals the binding behaviors of chlorogenic acid with five milk proteins and provides basic data that can help to clarify the binding mechanism.     

Interaction of Sulfadiazine with Model Water Soluble Proteins: A Combined Fluorescence Spectroscopic and Molecular Modeling Approach

The binding behavior of antibacterial drug sulfadiazine (SDZ) with water soluble globular proteins like bovine as well as human serum albumin (BSA and HSA, respectively) and lysozyme (LYS) was monitored by fluorescence titration and molecular docking calculations. The experimental data reveal that the quenching of the intrinsic protein fluorescence in presence of SDZ is due to the strong interaction in the drug binding site of the respective proteins. The Stern-Volmer plot shows positive deviation at higher quencher concentration for all the proteins and was explained in terms of a sphere of action model. The calculated fluorophore-quencher distances vary within 4?~?11 Å in different cases. Fluorescence experiments at different temperature indicate thermodynamically favorable binding of SDZ with the proteins with apparently strong association constant (~10⁴–10⁵ M^?1) and negative free energy of interaction within the range of ?26.0?~??36.8 kJ mol^?1. The experimental findings are in good agreement with the respective parameters obtained from best energy ranked molecular docking calculation results of SDZ with all the three proteins.     

Angular correlations and the tau-neutrino mass

Effects of a nonzero mass for the tau neutrinov _τ as well as a right-handed charged-current contribution to the τ?v _τ coupling are discussed. Angular correlations of the decay products of τ⁺τ^? ine ⁺ e ^? annihilation are calculated as functions of \(m_{v_\tau } \) and the relative amount of right-handed τ?v _τ coupling.     

Spectroscopic study and application of the interaction mechanism of meloxicam with trypsin

Abstract

In order to explore the interaction between meloxicam and trypsin, the interaction mechanism between meloxicam and trypsin was studied by fluorescence spectrum, UV-vis absorption spectrum, circular dichroism spectrum, and molecular docking simulation under the experimental condition of pH = 7.40. The results of spectral experiments showed that meloxicam could effectively quench the internal fluorescence of trypsin in the form of static quenching, formed a stable complex at 1:1, and changed the conformation of trypsin. The results of thermodynamic constant showed that ΔG?H?S?>?0 indicates that the main force type of the binding system was hydrophobic interaction and hydrogen bonding. Molecular docking technique showed that the best binding site between meloxicam and trypsin was near the catalytic active center of trypsin, and the interaction between them changed the microenvironment of amino acid residues in the catalytic active center of trypsin. The mathematical model of drug and protein showed that when the concentration ratio of meloxicam to trypsin was 1:1, the protein binding rate of the binding system was 5.15%. The concentration ratio of meloxicam to trypsin was 30: 1, and the protein binding rate was 45.4%. The results showed that when the drug concentration was high, the binding effect of the system had a great influence on the concentration of free trypsin.     

Reaction mechanism of tylosin tartrate with lysozyme

Under simulated physiological conditions, the interaction between tylosin tartrate and lysozyme was investigated at pH?=?7.40 by fluorescence spectroscopy. The results indicated that tylosin tartrate could strongly quench the intrinsic fluorescence of lysozyme. By determining the quenching constants of the reaction between tylosin tartrate and lysozyme at different temperatures, the quenching mechanism was proven to be a static quenching process. The thermodynamic parameters (ΔH°, ΔS°) of the reaction between tylosin tartrate and lysozyme were obtained by the Van’t Hoff equation, and were 27.80?kJ mol^?1 and 166.28?J mol^?1 K^?1, respectively. The results showed that hydrophobic interaction between tylosin tartrate and lysozyme was dominant. Synchronous fluorescence spectra revealed that the conformation of lysozyme was changed. This method could be applied to measure the content of tylosin tartrate.     

Dynamics of polyglutamic acids in α-helical and coil states. Comparison with dynamics of some globular proteins. Rayleigh scattering of Mössbauer radiation (RSMR) data

Summary The classical model system poly-L-glutamic acid (poly-Glu), was investigated in a disordered coil state (atpH=7.0) and in helix state (atpH=2.0) by the RSMR technique. By considering that the coil state of poly-Glu models unfolded (random coil) state and α-helix state models the fluctuating secondary structure (during consequent folding of protein), a comparative analysis of the dynamical properties of poly-Glu in different states with the dynamical properties of different proteins in the native state (α-helical myoglobin and HSA, partially β-sheet lysozyme) and in intermediate (molten globule) state (α-lactalbumin) was performed. This comparison brings some unpredicted results: native α-helical proteins behave close to random coil, native partially β-sheet proteins behave close to fluctuating secondary structure (α-helix) and the dynamic behaviour of molten-globule state (partially β-sheet α-lactalbumin) is not different from the behaviour of lysozyme and much more rigid than that of native α-helical proteins. Paper presented at ICAME-95, Rimini, 10–16 September 1995.     

The study of the binding energy of nuclear matter in the relativistic σ− ω− π model with Δ isobar degree of freedom

The relativistic σ?ω?π model is proposed and used to calculate the binding energy of relativistic nuclear matter. By coupling Δ isobar to the σ meson, the zero-point fluctuation energy of the Δ isobar in the one loop approximation is derived. We calculate the effective mass of nucleon and Δ isobar, exchange and correlation energies, pressure and incompressibility of nuclear matter. The density dependence correction to σNN ωNN coupling constants is a very important mechanism to saturate the binding energy. The pion propagator is nuclear matter is constructed by the relativistic particle-hole, delta-hole and short-range correlation. The pion dispersion relation is calculated we find it’s very sensitive to the effective mass of nucleon and Δ isobar.     

Quenchers Induce Wavelength Dependence on Protein Fluorescence Lifetimes

We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein’s aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher’s extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.     

Adsorption Kinetics and Binding Studies of Protein Quantum Dots Interaction: A Spectroscopic Approach

Protein Quantum dots interaction is crucial to investigate for better understanding of the biological interactions of QDs. Here in, the model protein Bovine serum albumin (BSA) was used to evaluate the process of protein QDs interaction and adsorption on QDs surface. The modified Stern-Volmer quenching constant (K_a), number of binding sites (n) at different temperatures (298 308 and 318 K?±?1) and corresponding thermodynamic parameters (ΔG?<?0, ΔH?<?0, and ΔS?>?0) were calculated. The quenching constant (K_s) and number of binding sites (n) is found to be inversely proportional to temperature. It signified that static quenching mechanism is dominant over dynamic quenching. The standard free energy change (ΔG?<?0) implies that the binding process is spontaneous, while the enthalpy change (ΔH?<?0) suggest that the binding of QDs to BSA is an enthalpy-driven process. The standard entropy change (ΔS?>?0) suggest that hydrophobic force played a pivotal role in the interaction process. The adsorption process were assessed and evaluated by pseudofirst-order, pseudosecond-order kinetic model, and intraparticle diffusion model.     

Interaction mechanism between 4-aminoantipyrine and the enzyme lysozyme

4-Aminoantipyrine (AAP) is scarcely administered as a kind of analgesic drug because of the side effect. The residue of AAP in the environment poses a potential threat to human health. To evaluate the toxicity of AAP at the protein level, the effects of AAP on lysozyme were investigated using spectroscopic and molecular docking methods. Addition of AAP effectively quenched the intrinsic fluorescence of lysozyme. Static quenching of lysozyme by AAP revealed the formation of complex. After the inner filter effect was eliminated, the number of binding sites, the binding constant and the thermodynamic parameters were measured, and indicated that AAP can spontaneously bind with lysozyme through hydrophobic interactions with one binding site. Molecular docking results revealed that AAP bound into the enzyme active site and interacted with the Trp 62 and Trp 63 residues of lysozyme, which illustrated that the lysozyme activity was inhibited by AAP, in accordance with the results of the lysozyme activity experiment. Furthermore, the binding of AAP can result in demonstrable change of the conformation of lysozyme. This work is helpful for clarifying the molecular toxic mechanism of AAP in vivo.     

Two-photon annihilation rate of the positronic HCN molecule

Positron binding by the HCN molecule was studied within a modified adiabatic approximation. This approach, in which the positron is treated as a light pseudonucleus with fractional charge, leads to the binding energy of 0.65?mHartree. Using effective positron mass which reproduces the Quantum Monte Carlo binding energy gives an estimation of the two-photon annihilation rate of e ⁺HCN, equal to 0.091?ns^?1.     

Structure and stability of self-assembled actin-lysozyme complexes in salty water

Interactions between actin, an anionic polyelectrolyte, and lysozyme, a cationic globular protein, have been examined using a combination of synchrotron small-angle x-ray scattering and molecular dynamics simulations. Lysozyme initially bridges pairs of actin filaments, which relax into hexagonally coordinated columnar complexes comprised of actin held together by incommensurate one-dimensional close-packed arrays of lysozyme macroions. These complexes are found to be stable even in the presence of significant concentrations of monovalent salt, which is quantitatively explained from a redistribution of salt between the condensed and the aqueous phases.     

Momentum space of constant curvature and the “rainbow” approximation in quantum field theory

The behavior of the mass operator is studied in “rainbow” graph approximation in the momentum space of constant curvature with the group of motions SO(4,1). The infrared divergences occuring there are eliminated by a multiplicative renormalization. When x?4ι ^?2 (whereι is the “fundamental length”), the resulting asymptotic (x ? m² _c) expressions for the mass operator Σ_R (x) and its imaginary part are analytic in the coupling constant at zero, while in the domain x?4ι ^?2 a logarithmic branching occurs, and the function grows linearly. The assumption that there are “superheavy particles” in nature (with m _c ² ?hι ^?2) in the asymptotic domain x?4ι _?2 leads to a violation of the positive definiteness for the imaginary part of the mass operator.     

Fluorescence Quenching Study on the Interaction between Quercetin and Lipoxygenase

The interaction between quercetin and lipoxygenase was investigated by fluorescence spectroscopy. The analysis of the emission quenching at different temperatures revealed that the quenching mechanism correspond to a static process and, as consequence, a complex quercetin-lipoxygenase is formed. The thermodynamic parameters ΔG, ΔH and ΔS were calculated to be?32.57 kJmol^?1,?3.21 kJmol^?1 and 87.14 Jmol^?1K^?1 respectively, which suggest that hydrophobic forces plays a major role in the stabilization of the complex quercetin-lipoxygenase. The distance, r, between donor (lipoxygenase) and acceptor (quercetin) was calculated to be 3.84 nm based on Förster’s non-radiative energy transfer theory. The results obtained from the evaluation of three dimensional florescence spectra suggest a conformational modification of the protein in the region of the coupling with quercetin.