Alcohol induced structural and dynamic changes in β-lactoglobulin in aqueous solution: A neutron scattering study

Structural and dynamic properties of β-lactoglobulin (β-LG) were revealed as a function of alcohol concentration in ethanol- and trifluoroethanol(TFE)-water mixtures with circular dichroism (CD), small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS). The CD spectra showed that an increase in TFE concentration promotes the formation of the β-sheet structure of β-LG. The SANS-intensities were fitted using form factors for two attached spheres for the native and native-like states of the protein. At higher alcohol concentrations, where aggregation takes place, a form factor modelling diffusion limited colloidal aggregation (DLCA) was employed. The QENS-data were analyzed in terms of internal motions for all alcohol concentrations. While low concentrations of TFE (10% (v/v)) lead to an increase of the mean square amplitudes of vibrations < u²> and a retention of a native-like structure — but not to an increase of the characteristic radius of proton diffusion processes a. Addition of 20% (v/v) of TFE induces aggregation, going along with a further increase of < u²>. Further increase of TFE concentration to 30% (v/v) changes the nanoscale structure of the oligomeric nucleate, but induces no further significant changes in < u²>. The present study underlines the necessity of methods sensitive to the dynamics of a system to obtain a complete picture of a molecular process.     

Using polarization analysis to separate the coherent and incoherent scattering from protein samples

Polarization analysis was used to separate experimentally the coherent and spin-incoherent nuclear static scattering functions, from a representative set of samples of interest for protein studies. This method had so far limited application in the study of amorphous materials, despite the relevance of the information that it provides. It allows, for instance, the experimental determination of the structure factor of materials containing a significant amount of hydrogen atoms, avoiding the contamination of measurements by a non-negligible incoherent background. Knowledge of the relative importance of the coherent and incoherent terms at different Q-values is also a pre-requisite for the interpretation of quasielastic neutron scattering experiments, performed at instruments in which the total dynamic scattering function is measured, such as conventional time-of-flight and backscattering spectrometers. Combining data from different instruments, it was possible to cover a wide Q-range, from the small-angle region (0.006 < Q < 0.04 Å^− 1) to the wide-angle region (up to ≈ 2.35 Å^− 1). Quantitative information was obtained on the fraction of coherent to spin-incoherent scattering from different protein samples: deuterated and protonated protein powders at different hydration levels and solutions of protonated proteins in D₂O at different concentrations. The results obtained are discussed in the context of the validity of the assumptions generally made when interpreting quasielastic neutron scattering experiments performed without polarization analysis.     

The impact of high hydrostatic pressure on structure and dynamics of β-lactoglobulin

Methods

Combining small-angle X-ray and neutron scattering measurements with inelastic neutron scattering experiments, we investigated the impact of high hydrostatic pressure on the structure and dynamics of β-lactoglobulin (βLG) in aqueous solution.

Background

βLG is a relatively small protein, which is predominantly dimeric in physiological conditions, but dissociates to monomer below about pH 3.

Results

High-pressure structural results show that the dimer–monomer equilibrium, as well as the protein–protein interactions, are only slightly perturbed by pressure, and βLG unfolding is observed above a threshold value of 3000 bar. In the same range of pressure, dynamical results put in evidence a slowing down of the protein dynamics in the picosecond timescale and a loss of rigidity of the βLG structure. This dynamical behavior can be related to the onset of unfolding processes, probably promoted from water penetration in the hydrophobic cavity.

General significance

Results suggest that density and compressibility of water molecules in contact with the protein are key parameters to regulate the protein flexibility.     

Biopolymer - Surfactant Interaction: 4 Kinetics of Binding of Cetyltrimethyl Ammonium Bromide with Gelatin,Hemoglobin, β-lactoglobulin and Lysozyme

Abstract

The binding of CTAB with the proteins, gelatin, hemoglobin, β-lactoglobulin and lysozyme follow first order kinetics and occurs either in two or three distinct stages. The number of stages depends on the overall configuration of the biopolymers. The denatured protein, gelatin has shown three-stage kinetics under all conditions, whereas the native proteins, hemoglobinn, β-lactoglobulin and lysozyme have exhibited two stage kinetics. Heat treated lysozyme in 8 mol dm-³ urea medium has also shown a two-stage kinetics. On the basis of non interacting binding sites on the proteins and independent sequential binding, the rates of reaction have been observed to increase with temperature and follow the trend k₁ >> k₂ > k₃. The interaction of CTA⁺ with the proteins is both electrostatic and hydrophobic. Hemoglobin has shown maximum reaction rate whereas, β-lactoglobulin has shown a minimum. The activation parameters for the kinetic process have exhibited almost non-variant Δ G? and Δ H? < T Δ S? The formation of activation complex in the Eyring model is entropy controlled so also the overall kinetics. An isokinetic entropy-enthalpy compensation phenomenon has been observed for the respective kinetic stages.     

Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

Dynamics of lysozyme and its hydration water under an electric field

The effects of a static electric field on the dynamics of lysozyme and its hydration water are investigated by means of incoherent quasi-elastic neutron scattering (QENS). Measurements were performed on lysozyme samples, hydrated respectively with heavy water (D ₂O) to capture the protein dynamics and with light water (H ₂O), to probe the dynamics of the hydration shell, in the temperature range from 210 < T < 260 K. The hydration fraction in both cases was about ～ 0.38 gram of water per gram of dry protein. The field strengths investigated were respectively 0 kV/mm and 2 kV/mm ( ～2 × 10 ⁶ V/m) for the protein hydrated with D ₂O and 0 kV and 1 kV/mm for the H ₂O-hydrated counterpart. While the overall internal protons dynamics of the protein appears to be unaffected by the application of an electric field up to 2 kV/mm, likely due to the stronger intra-molecular interactions, there is also no appreciable quantitative enhancement of the diffusive dynamics of the hydration water, as would be anticipated based on our recent observations in water confined in silica pores under field values of 2.5 kV/mm. This may be due to the difference in surface interactions between water and the two adsorption hosts (silica and protein), or to the existence of a critical threshold field value E _c ～2–3 kV/mm for increased molecular diffusion, for which electrical breakdown is a limitation for our sample.     

Probing the fatty acid binding site of β-lactoglobulins

The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10^?7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10^?7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpH's lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.     

Passiflin,a novel dimeric antifungal protein from seeds of the passion fruit

The intent was to isolate an antifungal protein from seeds of the passion fruit (Passiflora edulis) and to compare its characteristics with other antifungal proteins and bovine β-lactoglobulin in view of its N-terminal amino acid sequence similarity to β-lactoglobulin. The isolation procedure entailed ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on DEAE-cellulose, and FPLC-gel filtration on Superdex 75. The isolated 67-kDa protein, designated as passiflin, exhibited an N-terminal amino acid sequence closely resembling that of bovine β-lactoglobulin. It is the first antifungal protein found to have a β-lactoglobulin-like N-terminal sequence. Its dimeric nature is rarely found in antifungal proteins. It impeded mycelial growth in Rhizotonia solani with an IC₅₀ of 16 μM and potently inhibited proliferation of MCF-7 breast cancer cells with an IC₅₀ of 15 μM. There was no cross-reactivity of passiflin with anti-β-lactoglobulin antiserum. Intact β-lactoglobulin lacks antifungal and antiproliferative activities and is much smaller in molecular size than passiflin. However, it has been reported that hydrolyzed β-lactoglobulin shows antifungal activity. The data suggest that passiflin is distinct from β-lactoglobulin.     

Effect of bovine milk antigens and egg lysozyme on the binding of 59Fe-lactoferrin to platelet plasma membranes

• 1.1. Platelets bind specifically to lactoferrin. A significant similarity between human lactoferrin and some bovine milk proteins has been established.
• 2.2. Because of the structural homology of lactoferrin and cows milk proteins they are able to influence lactoferrins regulatory function on the level of its binding to membrane receptors on platelets.
• 3.3. An inhibitory effect of bovine α-lactalbumin and of β-lactoglobulin on lactoferrin-receptor interaction was shown.
• 4.4. Bovine α-lactalbumin competes with lactoferrin for the binding sites.
• 5.5. Scatchard plot analysis of data shows one binding site for lactoferrin in the presence of α-lactalbumin with an affinity constant, Ka = 0.46 × 10⁹ mol/1 and 335 receptors/cell.
• 6.6. The inhibitory effect of β-lactoglobulin reaches 62% and is different for the common fraction ⨿-lactoglobulin and the genetic variants β-lactoglobulin A and B.
• 7.7. β-lactoglobulin does not compete with lactoferrin for the membrane receptors.
• 8.8. Bovine casein and egg lysozyme stimulate ⁵⁹Fe-lactoferrin binding to the receptors. The mechanism of these effects is still unknown.
• 9.9. Tested alimentary antigens are able to interact with lactoferrin and also with some platelet membrane structures.
• 10.10. Established changes in lactoferrin binding to the platelet membrane might be in relation to lactoferrins regulatory function and (or) eliminating mechanisms of these alimentary antigens.

     

The protein-solvent glass transition

The protein dynamical transition and its connection with the liquid-glass transition (GT) of hydration water and aqueous solvents are reviewed. The protein solvation shell exhibits a regular glass transition, characterized by steps in the specific heat and the thermal expansion coefficient at the calorimetric glass temperature T_G ≈ 170 K. It implies that the time scale of the structural α-relaxation has reached the experimental time window of 1–100 s. The protein dynamical transition, identified from elastic neutron scattering experiments by enhanced amplitudes of molecular motions exceeding the vibrational level [1], probes the α-process on a shorter time scale. The corresponding liquid-glass transition occurs at higher temperatures, typically 240 K. The GT is generally associated with diverging viscosities, the freezing of long-range translational diffusion in the supercooled liquid. Due to mutual hydrogen bonding, both, protein- and solvent relaxational degrees of freedom slow down in paralled near the GT. However, the freezing of protein motions, where surface-coupled rotational and librational degrees of freedom are arrested, is better characterized as a rubber-glass transition. In contrast, internal protein modes such as the rotation of side chains are not affected. Moreover, ligand binding experiments with myoglobin in various glass-forming solvents show, that only ligand entry and exit rates depend on the local viscosity near the protein surface, but protein-internal ligand migration is not coupled to the solvent. The GT leads to structural arrest on a macroscopic scale due to the microscopic cage effect on the scale of the intermolecular distance. Mode coupling theory provides a theoretical framework to understand the microcopic nature of the GT even in complex systems. The role of the α- and β-process in the dynamics of protein hydration water is evaluated. The protein-solvent GT is triggered by hydrogen bond fluctuations, which give rise to fast β-processes. High-frequency neutron scattering spectra indicate increasing hydrogen bond braking above T_G.     

Hydrophobic thickness, lipid surface area and polar region hydration in monounsaturated diacylphosphatidylcholine bilayers: SANS study of effects of cholesterol and β-sitosterol in unilamellar vesicles

The influence of a mammalian sterol cholesterol and a plant sterol β-sitosterol on the structural parameters and hydration of bilayers in unilamellar vesicles made of monounsaturated diacylphosphatidylcholines (diCn:1PC, n = 14-22 is the even number of acyl chain carbons) was studied at 30 °C using small-angle neutron scattering (SANS). Recently published advanced model of lipid bilayer as a three-strip structure was used with a triangular shape of polar head group probability distribution (Ku?erka et al., Models to analyze small-angle neutron scattering from unilamellar lipid vesicles, Physical Review E 69 (2004) Art. No. 051903). It was found that 33 mol% of both sterols increased the thickness of diCn:1PC bilayers with n = 18-22 similarly. β-sitosterol increased the thickness of diC14:1PC and diC16:1PC bilayers a little more than cholesterol. Both sterols increased the surface area per unit cell by cca 12 Å² and the number of water molecules located in the head group region by cca 4 molecules, irrespective to the acyl chain length of diCn:1PC. The structural difference in the side chain between cholesterol and β-sitosterol plays a negligible role in influencing the structural parameters of bilayers studied.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Use of immobilized Streptomyces griseus proteases (pronase) as a probe of structural transitions of lysozyme, β-lactoglobulin and casein

The native - denatured (N U) structural transition in lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17), β-lactoglobulin and caseins have been studied by proteolysis using immobilized Streptomyces griseus proteases (pronase) as a probe. A diverse range of susceptibility to urea denaturation was revealed by evaluation of initial rates and pseudo first-order rate constants for hydrolysis of these proteins. Comparison of the rate of hydrolysis of lysozyme vis-à-vis performic acid oxidized-lysozyme showed that the degree of backbone accessibility for native lysozyme, even in concentrated urea solutions, was less than that of the oxidized protein. At pH 7.5, native lysozyme appeared to possess the most stable structure, followed by β-lactoglobulin and, finally, the caseins. It is postulated that the proteolytic rate depends upon accessibility of a susceptible bond(s) or subtle conformational changes in the least stable domain. Following cleavage of this bond(s), K_D increases thus exposing more backbone. Use of pronase immobilized on porous succinamidopropyl-glass beads resulted in increased enzyme stability and eliminated autolysis. Consequently, immobilized proteases are an excellent probe of structural transitions of protein substrates in denaturants.     

Time-resolved quasielastic neutron scattering studies of native photosystems

The internal molecular dynamics of proteins plays an important role in a number of functional processes in native photosystems. Prominent examples include the photocycle of bacteriorhodopsin and electron transfer in the reaction center of plant photosystem II. In this regard, the recently developed technique of time-resolved quasielastic neutron scattering with laser excitation opens up new perspectives for the study of protein/membrane dynamics in specific functional states of even complex systems. The first direct observation of a functionally modulated protein dynamics has just recently been reported for the model system bacteriorhodopsin (Pieper et al., Phys. Rev. Lett. 100, 2008, 228103.), where a transient softening of the protein was observed on a timescale of ∼ 1 ms along with the large-scale structural change in the M-intermediate of bacteriorhodopsin. In contrast, photosystem II membrane fragments with inhibited electron transfer show a suppression of protein dynamics ∼160 μs after the actinic laser flash (Pieper and Renger, Biochemistry 48, 2009, 6111). This effect may reflect aggregation-like conformational changes capable of dissipation of excess excitation energy to prevent photodamage in the absence of Q_A→Q_B electron transfer. These findings indicate that proteins exhibit a remarkable flexibility to accommodate different functional processes. This contribution will discuss methodical aspects, challenges, and recent applications of laser-excited, time-resolved quasielastic neutron scattering.     

Activation volumes of post protein reactions: The binding of bromophenol blue to β-lactoglobulin B

The binding of bromophenol blue to β-lactoglobulin B was studied in a high pressure temperature-jump apparatus. The process is characterized by a fast binding step with a positive volume change, followed by a slow isomerization with a comparatively small volume change. The spectral changes, pH dependence, and thermodynamic parameters of the binding indicate that the process is controlled by hydrophobic interactions between the dye and a site exposed to the solvent after the “N → R” transition of β-lactoglobulin has taken place.     

Incoherent neutron scattering of copper azurin: a comparison with molecular dynamics simulation results

The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range. Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample. Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions. The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample. Such an excess is reminiscent of the “boson peak” observed in glass-like materials. This vibrational peak is quite well reproduced by MD simulations, although at a lower energy. The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor. Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one. Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure. Received: 12 October 1998 / Revised version: 19 February 1999 / Accepted: 1 March 1999     

Computer analyses of characteristic infrared bands of globular proteins.

Infrared spectra of myoglobin, ribonuclease, lysozyme, α-chymotrypsin, α-lactalbumin, and β-lactoglobulin A were obtained in deuterium oxide solution in units of absorbance versus wavenumber from 1340 to 1750 cm^?1. The spectra were resolved into Gaussian components by means of an iterative computer program. Resolved characteristic absorption peaks for the two infrared active amide I′ components of antiparallel chain-pleated sheets (β-structure) were obtained. The characteristic amide I′ peaks of α-helical regions and apparently unordered regions overlap in D₂O solution. Absorptivity values for the resolved β-structure peak around 1630 cm^?1 were estimated on the basis of the known structure of ribonuclease, lysozyme, and β-chymotrypsin. The β-structure content of β-lactoglobulin was estimated to be ca. 48% of α-lactalbumin ca. 18%, and of α_s-casein close to zero. The results are in general agreement with conclusions drawn from circular dichroism and optical rotatory dispersion studies.     

Human Microtubule-Associated-Protein Tau Regulates the Number of Protofilaments in Microtubules: A Synchrotron X-Ray Scattering Study

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from αβ heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius 〈R^MT〉 of MTs with increasing Φ, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in 〈R^MT〉 seems to be isoform independent. Significantly, 〈R^MT〉 was observed to rapidly increase for 0 < Φ < 0.2 and saturate for Φ between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C_o^MT of MTs leading to changes in the curvature C^MT (=1/R^MT). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.     

Hydrolysis of β-lactoglobulin by thermolysin and pepsin under high hydrostatic pressure

Hydrolysis of β-lactoglobulin with thermolysin and pepsin at pressures ranging between 0.1 and 350 MPa showed a significant increase of cleavage rates. Pressure-induced changes of susceptibility to hydrolysis of β-lactoglobulin proteolytic sites were also observed. The pressure, raised to 200 MPa, accelerates the hydrolysis of β-lactoglobulin by thermolysin and changes obtained peptide profiles. Initially, higher pressure makes the N-terminal, and to a smaller extent, C-terminal peptide fragments of β-lactoglobulin molecule, more susceptible to removal by thermolysin. This indicates combined influence of pressure-induced thermolysin activation and partial unfolding of β-lactoglobulin by compression at neutral pHs. The rates of hydrolysis of β-lactoglobulin by pepsin (negligible at 0.1 MPa) are increased considerably with pressure up to 300 MPa. The Susceptibility of β-lactoglobulin proteolytic sites to peptic cleavage remains constant over all the studied pressure range. The lack of significant qualitative changes in the peptic peptide profiles produced at different pressures and at clearly pressure-dependent rates points to negative reaction volume changes as the major factor in peptic hydrolysis of β-lactoglobulin under high pressure. Thus the β-lactoglobulin molecule resists pressure-induced unfolding in acid pHs and yields to it in neutral pHs. © 1995 John Wiley & Sons, Inc.